Screening method and compounds for modulating telomerase activity

ABSTRACT

The present invention embraces methods for identifying compounds that modulate the activity of telomerase. Compounds of the invention are identified by designing or screening for a compound which binds to at least one amino acid residue of the TRBD, “thumb,” “finger,” and/or “palm” domain; or FP-pocket, PT-pocket or Th-pocket of telomerase and testing the compound for its ability to modulate the activity of telomerase. Compounds selected for interacting with the T-pocket or Fingers-Palm pocket of telomerase are also provided.

This application is a U.S. National Stage Application ofPCT/US2011/023959 filed Feb. 8, 2011 and is a continuation-in-partapplication claiming priority from U.S. patent application Ser. No.12/701,801 filed Feb. 8, 2010, U.S. patent application Ser. No.12/701,843 filed Feb. 8, 2010, and U.S. patent application Ser. No.12/701,947 filed Feb. 8, 2010, the contents of which are incorporatedherein by reference in their entireties.

BACKGROUND OF THE INVENTION

Any organism with linear chromosomes faces a substantial obstacle inmaintaining the terminal sequence of its DNA often referred to as the“end replication problem” (Blackburn (1984) Annu. Rev. Biochem.53:163-194; Cavalier-Smith (1974) Nature 250:467-470; Cech & Lingner(1997) Ciba Found. Symp. 211:20-34; Lingner, et al. (1995) Science269:1533-1534; Lundblad (1997) Nat. Med. 3:1198-1199; Ohki, et al.(2001) Mol. Cell. Biol. 21:5753-5766). Eukaryotic cells overcome thisproblem through the use of a specialized DNA polymerase, calledtelomerase. Telomerase adds tandem, G-rich, DNA repeats (telomeres) tothe 3′-end of linear chromosomes that serve to protect chromosomes fromloss of genetic information, chromosome end-to-end fusion, genomicinstability and senescence (Autexier & Lue (2006) Annu. Rev. Biochem.75:493-517; Blackburn & Gall (1978) J. Mol. Biol. 120:33-53;Chatziantoniou (2001) Pathol. Oncol. Res. 7:161-170; Collins (1996)Curr. Opin. Cell Biol. 8:374-380; Dong, et al. (2005) Crit. Rev. Oncol.Hematol. 54:85-93).

The core telomerase holoenzyme is an RNA-dependent DNA polymerase (TERT)paired with an RNA molecule (TER) that serves as a template for theaddition of telomeric sequences (Blackburn (2000) Nat. Struct. Biol.7:847-850; Lamond (1989) Trends Biochem. Sci. 14:202-204; Miller &Collins (2002) Proc. Natl. Acad. Sci. USA 99:6585-6590; Miller, et al.(2000) EMBO J. 19:4412-4422; Shippen-Lentz & Blackburn (1990) Science247:546-552). TERT is composed of four functional domains one of whichshares similarities with the HIV reverse transcriptase (RT) in that itcontains key signature motifs that are hallmarks of this family ofproteins (Autexier & Lue (2006) supra; Bryan, et al. (1998) Proc. Natl.Acad. Sci. USA 95:8479-8484; Lee, et al. (2003) J. Biol. Chem.278:52531-52536; Peng, et al. (2001) Mol. Cell 7:1201-1211). The RTdomain, which contains the active site of telomerase is thought to beinvolved in loose associations with the RNA template (Collins & Gandhi(1998) Proc. Natl. Acad. Sci. USA 95:8485-8490; Jacobs, et al. (2005)Protein Sci. 14:2051-2058). TERT however is unique, when compared toother reverse transcriptases in that it contains two domains N-terminalto the RT domain that are essential for function. These include the farN-terminal domain (TEN), which is the least conserved among phylogeneticgroups, but is required for appropriate human, yeast and ciliatedprotozoa telomerase activity in vitro and telomere maintenance in vivo(Friedman & Cech (1999) Genes Dev. 13:2863-2874; Friedman, et al. (2003)Mol. Biol. Cell 14:1-13). The TEN domain has both DNA- and RNA-bindingproperties. DNA-binding facilitates loading of telomerase to thechromosomes while RNA-binding is non-specific and the role of thisinteraction is unclear (Hammond, et al. (1997) Mol. Cell. Biol.17:296-308; Jacobs, et al. (2006) Nat. Struct. Mol. Biol. 13:218-225;Wyatt, et al. (2007) Mol. Cell. Biol. 27:3226-3240). A third domain, thetelomerase RNA binding domain (TRBD), is located between the TEN and RTdomains, and unlike the TEN-domain is highly conserved amongphylogenetic groups and is essential for telomerase function both invitro and in vivo (Lai, et al. (2001) Mol. Cell. Biol. 21:990-1000). TheTRBD contains key signature motifs (CP- and T-motifs) implicated in RNArecognition and binding and makes extensive contacts with stem I and theTBE of TER, both of which are located upstream of the template (Bryan,et al. (2000) Mol. Cell 6:493-499; Cunningham & Collins (2005) Mol.Cell. Biol. 25:4442-4454; Lai, et al. (2002) Genes Dev. 16:415-420; Lai,et al. (2001) supra; Miller, et al. (2000) supra; O'Connor, et al.(2005) J. Biol. Chem. 280:17533-17539). The TRBD-TER interaction isrequired for the proper assembly and enzymatic activity of theholoenzyme both in vitro and in vivo, and is thought to play animportant role (although indirect) in the faithful addition of multiple,identical telomeric repeats at the ends of chromosomes (Lai, et al.(2002) supra; Lai, et al. (2003) Mol. Cell 11:1673-1683; Lai, et al.(2001) supra).

Unlike TERT, TER varies considerably in size between species. Forexample, in Tetrahymena thermophila TER is only 159 nucleotides long(Greider & Blackburn (1989) Nature 337:331-337), while yeast harbors anunusually long TER of 1167 nucleotides (Zappulla & Cech (2004) Proc.Natl. Acad. Sci. USA 101:0024-10029). Despite the large differences insize and structure, the core structural elements of TER are conservedamong phylogenetic groups, suggesting a common mechanism of telomerereplication among organisms (Chen, et al. (2000) Cell 100:503-514; Chen& Greider (2003) Genes Dev. 17:2747-2752; Chen & Greider (2004) TrendsBiochem. Sci. 29:183-192; Ly, et al. (2003) Mol. Cell. Biol.23:6849-6856; Theimer & Feigon (2006) Curr. Opin. Struct. Biol.16:307-318). These include the template, which associates loosely withthe RT domain, and provides the code for telomere synthesis, and theTBE, which partly regulates telomerase's repeat addition processivity.In Tetrahymena thermophila, the TBE is formed by stem II and theflanking single stranded regions, and is located upstream and in closeproximity to the template (Lai, et al. (2002) supra; Lai, et al. (2003)supra; Licht & Collins (1999) Genes Dev. 13:1116-1125). Low-affinityTERT-binding sites are also found in helix IV and the templaterecognition element (TRE) of Tetrahymena thermophila TER.

TERT function is regulated by a number of proteins, some of which act bydirect association with the TERT/TER complex, while others act byregulating access of telomerase to the chromosome end through theirassociation with the telomeric DNA (Aisner, et al. (2002) Curr. Opin.Genet. Dev. 12:80-85; Cong, et al. (2002) Microbiol. Mol. Biol. Rev.66:407-425; Dong, et al. (2005) supra; Loayza & de Lange (2004) Cell117:279-280; Smogorzewska & de Lange (2004) Annu. Rev. Biochem.73:177-208; Smogorzewska, et al. (2000) Mol. Cell. Biol. 20:1659-1668;Witkin & Collins (2004) Genes Dev. 18:1107-1118; Witkin, et al. (2007)Mol. Cell. Biol. 27:2074-2083). For example, p65 in the ciliatedprotozoan Tetrahymena thermophila or its homologue p43 in Euplotesaediculatus, are integral components of the telomerase holoenzyme(Aigner & Cech (2004) RNA 10:1108-1118; Aigner, et al. (2003)Biochemistry 42:5736-5747; O'Connor & Collins (2006) Mol. Cell. Biol.26:2029-2036; Prathapam, et al. (2005) Nat. Struct. Mol. Biol.12:252-257; Witkin & Collins (2004) supra; Witkin, et al. (2007) supra).Both p65 and p43 are thought to bind and fold TER, a process requiredfor the proper assembly and full activity of the holoenzyme. In yeast,recruitment and subsequent up regulation of telomerase activity requiresthe telomerase-associated protein Est1 (Evans & Lundblad (2002) Genetics162:1101-1115; Hughes, et al. (1997) Ciba Found. Symp. 211:41-52;Lundblad (2003) Curr. Biol. 13:R439-441; Lundblad & Blackburn (1990)Cell 60:529-530; Reichenbach, et al. (2003) Curr. Biol. 13:568-574;Snow, et al. (2003) Curr. Biol. 13:698-704). Est1 binds the RNAcomponent of telomerase, an interaction that facilitates recruitment ofthe holoenzyme to the eukaryotic chromosome ends via its interactionwith the telomere binding protein Cdc13 (Chandra, et al. (2001) GenesDev. 15:404-414; Evans & Lundblad (1999) Science 286:117-120; Lustig(2001) Nat. Struct. Biol. 8:297-299; Pennock, et al. (2001) Cell104:387-396).

How telomerase and associated regulatory factors physically interact andfunction with each other to maintain appropriate telomere length isunder investigation. Structural and biochemical characterization ofthese factors, both in isolation and complexed with one another, can beused to determine how the interaction of the TRBD domain with stem I andthe TBE of TER facilitate the proper assembly and promote the repeataddition processivity of the holenzyme.

While in vitro and in vivo screening assays have been developed toidentify agents which modulate telomerase activity or telomere binding,focus has not been placed on identifying agents with a degree ofspecificity for particular domains or substrate pockets. See, U.S. Pat.Nos. 7,067,283; 6,906,237; 6,787,133; 6,623,930; 6,517,834; 6,368,789;6,358,687; 6,342,358; 5,856,096; 5,804,380; and 5,645,986 and US2006/0040307.

SUMMARY OF THE INVENTION

In one embodiment, the present invention features a compound whichinteracts with the T-pocket or fingers-palm (FP) pocket of telomerase.In another embodiment, the invention includes a compound having astructure of Formula I, Formula II, Formula III or Formula IV. In yetanother embodiment, the invention is a compound as listed in Table 8,Table 9, Table 10 or Table 11.

Pharmaceutical compositions and methods for using the compounds of theinvention to inhibit or stimulate telomerase activity, and in theprevention or treatment of a disease or disorder associated withtelomerase are also provided. Diseases or disorders embraced by theinvention include, cancer, Alzheimer's disease, Parkinson's disease,Huntington's disease, stroke, osteoporosis, diabetes and age-relatedmacular degeneration.

The present invention also features methods for identifying a compoundwhich modulates the activity of telomerase. The methods of thisinvention involve, (a) designing or screening for a compound which bindsto at least one amino acid residue of the TRBD domain, “thumb” domain,“palm” domain, “finger” domain, Motif 2, Motif B′, Thumb loop or Thumbhelix domains; and (b) testing the compound designed or screened for in(a) for its ability to modulate the activity of telomerase, therebyidentifying a compound that modulates the activity of telomerase. In oneembodiment, the TRBD domain of telomerase contains the amino acidresidues set forth in Table 1. In another embodiment, the “thumb,”“palm” and/or “finger” domain contains the amino acid residues set forthin Table 2. In other embodiments, step (a) is carried out in silico.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of telomerase (TERT). FIG. 1A shows theprimary of human, yeast and Tetrahymena thermophila TERT showing thefunctional domains and conserved motifs. FIG. 1B is the primarystructure and conserved motifs of the Tribolium castaneum TERT. FIG. 1Cshows TERT domain organization with the RNA-binding domain (TRBD), thereverse transcriptase domain composed of the “fingers” and “palm”subdomains, and the “thumb” domain depicted.

FIGS. 2A and 2B show a sequence alignment and schematic of secondarystructure of Tetrahymena thermophila TRBDs (TETTH; SEQ ID NO:1) comparedwith the TRBDs from ciliated protozoa such as, Euplotes aediculatus(EUPAE; SEQ ID NO:2) and Oxytricha trifallax (OXYTR; SEQ ID NO:3);mammals such as human (SEQ ID NO:4) and mouse (SEQ ID NO:5); fungi suchas Schizosaccharomyces pombe (SCHPO; SEQ ID NO:6) and Saccharomycescerevisiae (YEAST; SEQ ID NO:7); and plants such as Arabidopsis thaliana(ARATH; SEQ ID NO:8) produced by ALSCRIPT Barton (1993) Protein Eng.6:37-40). Conserved residues in key signature motifs are indicated andmutated residues that affect RNA-binding and telomerase function arealso indicated. The solid triangles define the boundaries of the TRBDconstruct used in the studies herein.

FIGS. 3A-3C show the sequence alignment and surface conservation ofTribolium castaneum TERT (TRICA; SEQ ID NO:9) compared with TERTs fromvarious phylogenetic groups including mammals such as mouse (SEQ IDNO:10) and human (SEQ ID NO:11); plants such as Arabidopsis thaliana(ARATH; SEQ ID NO:12); fungi such as Saccharomyces cerevisiae (YEAST;SEQ ID NO:13) and Schizosaccharomyces pombe (SCHPO; SEQ ID NO:14); andprotozoa such as Tetrahymena thermophila (TETTH; SEQ ID NO:15) andEuplotes aediculatus (EUPAE; SEQ ID NO:16) produced by ClustalW2 (Larkinet al. (2007) Bioinformatics 23:2947-2948). Conserved residues in keysignature motifs are indicated. K210 of helix α10 and polar residues(K406, K416, K418, N423) of the “thumb” domain implicated in directcontacts with the backbone of the DNA substrate are also shown.

FIG. 4 is a schematic of the primary structure of the RNA component(TER) of telomerase from Tetrahymena thermophila. Stem I, TBE and thetemplate are indicated.

FIG. 5 depicts TERT RNA template associations. The nucleotide located atthe 5′-end of the RNA template (rC1) is coordinated by Ile196 and Val197of motif 2 and Gly309 of motif B′. rU2 interacts with Pro311 of motif B′and rG3 coordinates the backbone of helix α15 via a water molecule(Wat18).

FIG. 6 depicts TERT telomeric DNA associations. Interactions between thethumb loop and the DNA are mostly backbone and solvent mediated. Also,the side chains of Lys416 and Asn423 that form part of the thumb loopextend toward the center of the ring and coordinate the DNA backbone.

FIG. 7 depicts DNA interactions with the primer grip region and theactive site. Shown is a stereo view of the DNA interactions with motif Eand the active site residues. The tip of the primer grip region (loopshown on left), formed by the backbone of residues Cys390 and Gly391abuts the ribose group of C22 and this interaction guides the 3′-end ofthe DNA at the active site of the enzyme for nucleotide addition. Theactive site bound magnesium ion (sphere) coordinates the DNA backboneformed by the last two nucleotides. The nucleotide binding pocket ofTERT, which is partially occupied by the last DNA nucleotide, is in partformed by the highly conserved residue Val342 and the invariant Tyr256and Gly308.

FIG. 8 shows the telomerase T-pocket and drug binding. Conserved motifsT, 1 and 2 that form the T-pocket are shaded.

FIG. 9 illustrates the mode of inhibition of telomerase by TERTmodulators. The two nucleotides bound at the active site of telomeraseare shown. The small molecule inhibitor of telomerase clashing with oneof the nucleotides is also shown.

DETAILED DESCRIPTION OF THE INVENTION

Telomerase, a ribonucleoprotein complex, replicates the linear ends ofeukaryotic chromosomes, thus taking care of the “end of replicationproblem”. TERT contains an essential and universally conserved domain(TRBD; FIG. 1) that makes extensive contacts with the RNA (TER)component of the holoenzyme and this interaction facilitates TERT/TERassembly and repeat addition processivity. The TRBD domain is highlyconserved among phylogenetic groups and is essential for the function oftelomerase. Extensive biochemical and mutagenesis studies have localizedTRBD binding to stem I and the TEB, interactions that are thought to beimportant for the proper assembly and stabilization of the TERT/TERcomplex as well as the repeat addition processivity of the holoenzyme.The atomic structure of the TRBD domain has now been identified, therebyproviding information about TERT/TER binding. The RNA-binding site ofTRBD is an extended groove on the surface of the protein that is partlyhydrophilic and partly hydrophobic in nature and is formed by thepreviously identified T- and CP-motifs shown to be important fortelomerase function. The size, organization and chemical nature of thisgroove indicates that the TRBD domain interacts with both double- andsingle-stranded nucleic acid, possibly stem I or II and the ssRNA thatconnects them.

In addition to the structure of the TRBD domain, it has now been shownthat three highly conserved domains, TRBD, the reverse transcriptase(RT) domain, and the C-terminal extension thought to represent theputative “thumb” domain of TERT, are organized into a ring-likestructure (FIG. 1C) that shares common features with retroviral reversetranscriptases, viral RNA polymerases and B-family DNA polymerases.Domain organization places motifs implicated in substrate binding andcatalysis in the interior of the ring, which can accommodateseven-to-eight bases of double stranded nucleic acid. Modeling of anRNA/DNA heteroduplex in the interior of this ring reveals a perfect fitbetween the protein and the nucleic acid substrate and positions the3′-end of the DNA primer at the active site of the enzyme providingevidence for the formation of an active telomerase elongation complex.

Indeed, as the high-resolution structure of the telomerase catalyticsubunit, TERT, in complex with its RNA-templating region, part of thecomplementary DNA sequence, nucleotides and magnesium ions shows, theRNA-DNA hybrid adopts an A-form helical structure and is docked in theinterior cavity of the TERT ring. Protein-nucleic acid associationsinduce global TERT conformational changes that lead to a decrease in thediameter of the interior cavity of the ring facilitating the formationof a tight telomerase elongation complex. The enzyme, which uses atwo-metal binding mechanism for nucleotide association and catalysis,contains two nucleotides at its active site. One of the nucleotides isin position for attack by the 3′-hydroxyl of the incoming DNA primerwhile the second site appears to hold the nucleotide transiently,allowing for RNA template-dependent selectivity and telomeraseprocessivity.

The TRBD domain, as well as RT and “thumb” domains, are highly conserveddomains among phylogenetic groups. As such, these domains serve as idealcandidates for telomerase inhibitors. In this regard, telomerase is anideal target for treating human diseases or conditions relating tocellular proliferation and senescence.

Accordingly, the present invention relates to the use of thehigh-resolution structure of Tetrahymena thermophila and Triboliumcastaneum telomerases for the identification of effector molecules thatmodulate the activity of telomerase. The term “effector” refers to anyagonist, antagonist, ligand or other agent that affects the activity oftelomerase. Effectors include, but are not limited to, peptides,carbohydrates, nucleic acids, lipids, fatty acids, hormones, organiccompounds, and inorganic compounds. The information obtained from thecrystal structures herein reveal detailed information which is useful inthe design, isolation, screening and determination of potentialcompounds which modulate the activity of telomerase. Compounds that bindthe TRBD domain and, e.g., sterically block TER binding or block RNPassembly act as effective telomerase-specific inhibitors, whereascompounds that mimic or facilitate TER binding or RNP assembly act aseffective telomerase-specific activators. Compounds that bind and blockthe active site or nucleotide binding site can also modulate telomeraseactivity. Similarly, compounds that interact with one or more amino acidresidues of telomerase in direct contact with DNA can block DNA bindingand act as effective telomerase-specific inhibitors, whereas compoundsthat mimic DNA act as effective telomerase-specific activators.

The term “effector” refers to an agonist, antagonist, ligand or otheragent that affects the activity of telomerase. Effectors that bind theT-pocket of the TRBD domain and, e.g., sterically block TER binding orblock RNP assembly act as effective telomerase-specific inhibitors,whereas effectors that mimic or facilitate TER binding or RNP assemblyact as effective telomerase-specific activators. Similarly, Effectorsthat bind the FP-pocket of telomerase and, e.g., occlude the active siteof telomerase act as effective telomerase-specific inhibitors, whereaseffectors in close proximity to the active site of the enzyme withoutoccluding the active site act as effective telomerase-specificactivators.

Molecules or compounds of the present invention were selected forinteracting with motifs T, 1, and/or 2 of the T-pocket or FP-pocket oftelomerase. Such selection was based upon various heterogeneousinteractions between the compound and telomerase including, but notlimited to van der Waals contacts, hydrogen bonding, ionic interactions,polar contacts, or combinations thereof. In this regard, the terms“bind,” “binding,” “interact,” or “interacting” are used interchangeablyherein to describe the physical interactions between amino acid residuesof motifs T, 1 and/or 2 of telomerase (See FIG. 9) or amino acidresidues of the FP-pocket and effectors thereof. In general, it isdesirable that the compound interacts with 2, 3, 4, 5, 6 or more of theamino acid residues of a domain disclosed herein to enhance thespecificity of the compound for one or more telomerase proteins.Accordingly, it is desirable that the compound interacts with between 2and 20, 2 and 10, or 2 and 5 of one or more of the domains or pocketsdisclosed herein.

The effector molecules of the invention have a wide variety of uses. Forexample, it is contemplated that telomerase modulators will be effectivetherapeutic agents for treatment of human diseases or conditions.Screening for agonists provides for compositions that increasetelomerase activity in a cell (including a telomere-dependentreplicative capacity, or a partial telomerase activity). Such agonisticcompositions provide for methods of immortalizing otherwise normaluntransformed cells, including cells which can express useful proteins.Such agonists can also provide for methods of controlling cellularsenescence. Conversely, screening for antagonist activity provides forcompositions that decrease telomere-dependent replicative capacity,thereby mortalizing otherwise immortal cells, such as cancer cells.Screening for antagonistic activity provides for, compositions thatdecrease telomerase activity, thereby preventing unlimited cell divisionof cells exhibiting unregulated cell growth, such as cancer cells. Ingeneral, the effector molecules of the invention can be used whenever itis desired to increase or decrease telomerase activity in a cell ororganism.

Broadly, the methods of the invention involve designing or screening fora test compound which binds to at least one amino acid residue of anessential telomerase domain or pocket disclosed herein; and testing thecompound designed or screened for its ability to modulate the activityof telomerase. In certain embodiments, the method of the presentinvention is carried out using various in silico, in vitro and/or invivo assays based on detecting interactions between one or more domainsor domain residues; or pockets or pocket residues of telomerase and atest compound.

In the context of the present invention, telomerase refers to a familyof enzymes which maintain telomere ends by addition of the telomererepeat TTAGGG. Telomerases are described, e.g., by Nakamura, et al.(1997) Science 277(5328):955-9 and O'Reilly, et al. (1999) Curr. Opin.Struct. Biol. 9(1):56-65. Exemplary telomerase enzymes of use inaccordance with the present invention are set forth herein in SEQ IDNOs:1-16 (FIGS. 2A-2B and FIGS. 3A-3C) and full-length sequences fortelomerase enzymes are known in the art under GENBANK Accession Nos.AAC39140 (Tetrahymena thermophila), NP_(—)197187 (Arabidopsis thaliana),NP_(—)937983 (Homo sapiens), CAA18391 (Schizosaccharomyces pombe),NP_(—)033380 (Mus musculus), NP_(—)013422 (Saccharomyces cerevisiae),AAC39163 (Oxytricha trifallax), CAE75641 (Euplotes aediculatus) andNP_(—)001035796 (Tribolium castaneum). For the purposes of the presentinvention, reference to telomerase refers to allelic and syntheticvariants of telomerase, as well as fragments of telomerase. Syntheticvariants include those which have at least 80%, preferably at least 90%,homology to a telomerase disclosed herein. More preferably, suchvariants correspond to the sequence of a telomerase provided herein, buthave one or more, e.g., from 1 to 10, or from 1 to 5 substitutions,deletions or insertions of amino acids. Fragments of telomerase andvariants thereof are preferably between 20 and 500 amino acid residuesin length or between 50 and 300 amino acids in length. An exemplaryfragment includes the approximately 250 amino acid residues encompassingthe TRBD domain of telomerase. Other fragments include the “thumb”domain and the reverse transcriptase domain and its subdomains, i.e.,the “finger” and “palm” subdomains. As depicted in FIG. 1A and FIGS. 2Aand 2B, the TRBD domain encompasses amino acid residues at or about254-519 of T. thermophila telomerase. As depicted in FIG. 1B and FIGS.3A-3C, the reverse transcriptase domain encompasses amino acid residuesat or about 160-403 of T. castaneum telomerase, and the “thumb” domainencompasses amino acid residues at or about 404-596 of T. castaneumtelomerase. Based upon the amino acid sequence comparisons depicted inFIGS. 2A, 2B, and 3A-3C, suitable domains and fragments of telomerasesfrom other species can be readily obtained based upon the location ofequivalent amino acid residues in a telomerase from other species.

The nearly all-helical structure of TRBD provides a nucleic acid bindingfold suitable for TER binding. An extended pocket on the surface of theprotein, formed by two conserved motifs (CP- and T-motifs) providesTRBD's RNA-binding pocket. The width and the chemical nature of thispocket indicate that it binds both single- and double-stranded RNA,likely stem I and the template boundary element (TBE). As disclosedherein, the structure of T. thermophila telomerase identified key aminoacid residues involved in RNP assembly of telomerase and the interactionbetween telomerase TRBD and TER. Accordingly, the present inventionembraces a compound, which binds to at least one amino acid residue ofthe TRBD domain. Essential amino acid residues of this domain areprovided in Table 1. The location of these residues in telomerases fromother organisms is also listed in Table 1. In particular embodiments, acompound of the invention binds to one or more of the amino acidresidues listed in Table 1, thereby modulating the activity oftelomerase.

TABLE 1 Essential TRBD Location in telomerases of other organismsResidues* Tt ^(#) Tc ^(†) At ^(#) Sp ^(#) Hs ^(#) Mm ^(#) Sc ^(#) Ot^(#) Ea ^(#) T-motif* F476 F233 Y134 F265 F185 F246 F231 F186 F202 F206Y477 Y234 Y135 Y266 Y186 Y247 Y232 Y187 Y203 Y207 T479 T236 P137 T268T188 T249 T234 T189 T205 T209 E480 E237 I138 E269 E189 E250 E235 E190E206 E210 Y491 Y248 I149 Y280 F200 Y261 Y246 F200 Y217 Y221 R492 R249R150 R281 R201 R262 R247 R201 R218 R222 K493 K250 K151 K282 K202 K263K248 H202 K219 L223 W496 W253 Y154 W285 W205 W266 W251 W205 W222 W226CP-motif* F323 F80 L36 L95 L55 Y90 Y90 Y58 F55 F59 L327 L84 K39 L99 Y59L94 L94 L62 L59 L63 K328 K85 H40 D100 N60 K95 R95 N63 S60 T64 K329 K86K41 K101 H61 T96 S96 S64 K61 K65 C331 C88 K43 C103 C63 C98 C98 C66 C63C67 L333 L90 P45 L108 — L100 — — L65 L69 P334 P91 V46 Q109 — R101 — —P66 P70 QFP-motif* Q375 Q132 Q47 Q158 Q83 Q145 Q130 R87 Q103 C107 I376I133 I48 V159 V84 V146 V131 V88 I104 V108 L380 L137 L52 I163 L88 V150L135 I92 L108 I112 I383 I140 I55 I166 I91 C153 C138 I95 F111 F115 I384I141 I56 C167 L92 L154 L139 L96 V112 F116 C387 C144 — I170 V95 L157 V142L99 V115 I119 V388 V145 — V171 F96 V158 V143 L100 F116 L120 P389 P146P57 P172 P97 P159 S144 P101 P117 P121 L392 L149 Y60 L175 I100 L162 L147M104 F120 F124 L393 L150 F61 L176 W101 W163 W148 F105 L121 L125 N397N154 N66 Q181 I106 N168 N153 N110 N125 N129 L405 L162 V74 I189 L114 T176L161 L118 M133 V137 F408 F165 I77 F192 F117 F179 F164 L121 F136 Y140Y422 Y179 L91 F206 L131 L193 L178 L135 L150 L154 I423 I180 H92 L207 M132T194 M179 L136 L151 L155 M426 M183 Y95 V210 I135 M197 M182 L139 F154I158 W433 W190 W102 F217 W142 W204 W189 W146 W161 W165 F434 F191 L103F218 L143 L205 L190 L147 L162 M166 Tt, Tetrahymena thermophila; At,Arabidopsis thaliana; Hs, Homo sapiens; Sp, Schizosaccharomyces pombe;Mm, Mus musculus; Sc, Saccharomyces cerevisiae; Tc, Tribolium castaneum;Ot, Oxytricha trifallax and Ea, Euplotes aediculatus. *Location is withreference to the full-length T. thermophila telomerase. ^(#)Location iswith reference to the telomerase sequences depicted in FIGS. 2A and 2B,i.e., SEQ ID NOs: 1-8. ^(†)Location is with reference to the telomerasesequence depicted in FIGS. 3A-3C.

As further disclosed herein, the structure of T. castaneum telomeraseidentified key amino acid residues of the reverse transcriptase and“thumb” domains. In particular, key amino acid residues of thenucleotide binding pocket were identified as well as amino acid residueswhich appear to make direct contacts with the backbone of the DNAsubstrate. Accordingly, the present invention also embraces a compound,which binds to at least one amino acid residue of the nucleotide bindingpocket of telomerase or residues which make direct contact with DNA.These residues are found in the “palm” and “finger” subdomains of thereverse transcriptase domain and the “thumb” domain of T. castaneumtelomerase. Essential amino acid residues of these domains are providedin Table 2. The location of these amino acid residues in various speciesis also listed in Table 2.

TABLE 2 Domain Location in telomerases of other organisms* Residues* TtAt Sp Hs Mm Sc Ea “Palm” K210 R573 K644 K547 N666 N656 E483 T558 V250L617 A690 I589 V711 A704 F529 M602 D251 D618 V691 D590 D712 D705 D530D603 I252 I619 D692 I591 V713 V706 V531 I604 A255 C622 A695 C594 A716A706 C534 C607 Y256 Y623 F696 Y595 Y717 Y707 Y535 Y608 G257 D624 D697D596 D718 D708 D536 D609 G305 G770 G801 G703 G830 G823 G629 G734 L306I771 I802 I704 I831 I824 L630 I735 L307 P772 P803 P705 P832 P825 F631P736 Q308 Q773 Q804 Q706 Q833 Q826 Q632 Q737 G309 G774 H805 G707 G834G827 G633 G738 V342 T814 I859 V741 V867 V860 A669 T780 D343 D815 D860D742 D868 D861 D670 D781 D344 D816 D861 D743 D869 D862 D671 D782 Y345Y817 Y862 F744 F870 F863 L672 Y783 F346 L818 L863 L745 L871 L864 F673L784 F347 F819 F864 F746 L872 L865 I674 L785 C348 I820 V865 I747 V873V866 I675 I786 S349 S821 S866 T748 T874 T867 S676 T787 N369 N846 N891S773 N899 N892 N701 N812 K372 K849 K894 K776 K902 K895 K704 K815 T373I850 F895 T777 T903 T896 I705 L816 “Finger” L184 L533 F612 I502 L621L611 M438 L514 N185 R534 R613 R503 R622 R612 R439 R515 I186 I535 F614L504 F623 F613 I440 L516 I187 I536 L615 L505 I624 I614 I441 I517 P188P537 P616 P506 P625 P615 P442 P518 K189 K538 K617 K507 K626 K616 K443K519 F193 F542 V621 F511 L630 L620 N447 F523 R194 R543 R622 R512 R631R621 E448 R524 A195 P544 M623 L513 P632 P622 F449 P525 I196 I545 V624I514 I633 I623 R450 I526 V197 M546 L625 T515 V634 V624 I451 M527 “Thumb”K406 Q888 T937 P815 S943 S936 S729 N860 K416 T898 T947 T825 S953 S946K739 T869 K418 N900 S949 S826 T955 T948 S741 N871 N423 K906 K955 H832K961 K954 R746 K877 Tt, Tetrahymena thermophila; At, Arabidopsisthaliana; Hs, Homo sapiens; Sp, Schizosaccharomyces pombe; Mm, Musmusculus; Sc, Saccharomyces cerevisiae;; and Ea, Euplotes aediculatus.*Location is with reference to the Tribolium castaneum telomerasesequence depicted in FIGS. 3A-3C.

In one embodiment, the compound interacts with one or more essentialamino acids of the QFP-motif, T-motif or CP-motif. In anotherembodiment, the compound interacts with one or more essential aminoacids of the T-motif and CP-motif. In a further embodiment, the compoundinteracts with one or more essential amino acids as set forth inTable 1. In particular embodiments, the compound interacts with between2 and 33 amino acid residues of the TRBD domain. In a particularembodiment, the compound interacts with one or more essential amino acidresidues set forth in Table 1, which have not been previously identifiedby mutation to affect RNA-binding and telomerase activity.

In another embodiment of the invention, the compound interacts with oneor more essential amino acids of the nucleotide binding pocket. In afurther embodiment, the compound interacts with one or more essentialamino acids of telomerase in direct contact with DNA. In yet a furtheressential amino acids as set forth in Table 2. In a particularembodiment, the compound interacts with one or more essential amino acidresidues set forth in Table 2, which have not been previously identifiedby mutation to affect nucleotide binding, DNA binding or telomeraseactivity. In particular embodiments, the compound interacts with between2 and 34 amino acid residues of the finger and palm domains. In specificembodiments, the compound binds to one or more of the amino acidresidues selected from the group of K189, R194, Y256, Q308, V342, andK372 of T. castaneum telomerase or their equivalents in other species.In other embodiments, the compound interacts with between 2 and 15 aminoacid residues of the palm and thumb domains. In specific embodiments,the compound binds to one or more of the amino acid residues selectedfrom the group of K210, K406, K416, K418, or N423 of T. castaneumtelomerase or their equivalents in other species.

As further disclosed herein, the structure of T. castaneum TERT incomplex with its RNA-templating region, part of the complementary DNA,nucleotides and magnesium ions identified key amino acid residues ofvarious domains which coordinate to place substrates in proximity withthe active site (D251, D343 and D344) of telomerase. These key aminoacid residues are located in Motif 2, Motif B′, Thumb Loop, and ThumbHelix regions of telomerase (FIG. 3). Accordingly, the present inventionfurther embraces a compound, which binds to at least one amino acidresidue of the Motif 2, Motif B′, Thumb Loop, or Thumb Helix oftelomerase. Amino acid residues of Motif 2 and Motif B′ are selectedfrom the group of Ile196, Val197, Gly309 and Pro311 of T. castaneumtelomerase or their equivalents in other species. Amino acid residues ofthe Thumb Loop and Thumb Helix are selected from the group of Tyr256,Gln308, Val342, Cys390, Gly391, Lys416, and Asn423 of T. castaneumtelomerase or equivalent amino acid residues thereof in a telomerasefrom another species.

In accordance with the present invention, molecular design techniquescan be employed to design, identify and synthesize chemical entities andcompounds, including inhibitory and stimulatory compounds, capable ofbinding to one or more amino acids of telomerase. The structure of thedomains of telomerase can be used in conjunction with computer modelingusing a docking program such as GRAM, DOCK, HOOK or AUTODOCK (Dunbrack,et al. (1997) Folding& Design 2:27-42) to identify potential modulatorsof telomerase proteins. This procedure can include computer fitting ofcompounds to domains or pockets disclosed herein to, e.g., ascertain howwell the shape and the chemical structure of the compound willcomplement the TRBD domain; or to compare the compound with the bindingof TER in the TRBD; or compare the compound with the binding of a DNAmolecule to the “thumb” domain; compare the compound with binding of anucleotide substrate to the nucleotide binding pocket; or compare thecompound to binding of TERT substrates in the Motif 2, Motif B′, ThumbLoop, and Thumb Helix. Computer programs can also be employed toestimate the attraction, repulsion and stearic hindrance of thetelomerase protein and effector compound. Generally, the tighter thefit, the lower the stearic hindrances, the greater the attractiveforces, and the greater the specificity, which are important featuresfor a specific effector compound which is more likely to interact withthe telomerase protein rather than other classes of proteins. In so faras the present invention has identified the amino acid residuesspecifically involved in substrate binding, the present invention offersspecificity not heretofore possible with conventional screening assays.

Alternatively, a chemical-probe approach can be employed in the designof telomerase modulators or effectors. For example, Goodford ((1985) J.Med. Chem. 28:849) describes several commercial software packages, suchas GRID (Molecular Discovery Ltd., Oxford, UK), which can be used toprobe the telomerase domains with different chemical probes, e.g.,water, a methyl group, an amine nitrogen, a carboxyl oxygen, and ahydroxyl. Favored sites for interaction between these regions or sitesof the telomerase domains and each probe are thus determined, and fromthe resulting three-dimensional pattern of such regions or sites aputative complementary molecule can be generated.

The compounds of the present invention can also be designed by visuallyinspecting the three-dimensional structure of the telomerase domains todetermine more effective inhibitors or activators. This type of modelingis generally referred to as “manual” drug design. Manual drug design canemploy visual inspection and analysis using a graphics visualizationprogram such as “O” (Jones, et al. (1991) Acta Crystallographica SectionA A47:110-119).

Initially effector compounds are selected by manual drug design. Thestructural analog thus designed can then be modified by computermodeling programs to better define the most likely effective candidates.Reduction of the number of potential candidates is useful as it may notbe possible to synthesize and screen a countless number of compoundvariations that may have some similarity to known inhibitory molecules.Such analysis has been shown effective in the development of HIVprotease inhibitors (Lam, et al. (1994) Science 263:380-384; Wlodawer,et al. (1993) Ann. Rev. Biochem. 62:543-585; Appelt (1993) Perspectivesin Drug Discovery and Design 1:23-48; Erickson (1993) Perspectives inDrug Discovery and Design 1:109-128). Alternatively, random screening ofa small molecule library could lead to modulators whose activity maythen be analyzed by computer modeling as described above to betterdetermine their effectiveness as inhibitors or activators.

Programs suitable for searching three-dimensional databases includeMACCS-3D and ISIS/3D (Molecular Design Ltd, San Leandro, Calif.),ChemDBS-3D (Chemical Design Ltd., Oxford, UK), and Sybyl/3 DB Unity(Tripos Associates, St Louis, Mo.). Programs suitable for compoundselection and design include, e.g., DISCO (Abbott Laboratories, AbbottPark, Ill.), Catalyst (Bio-CAD Corp., Mountain View, Calif.), andChemDBS-3D (Chemical Design Ltd., Oxford, UK).

The compounds designed using the information of the present inventioncan bind to all or a portion of the TRBD domain, nucleotide bindingdomain, “thumb” domain, Motif 2, Motif B′, Thumb Loop, and/or ThumbHelix of telomerase and may be more potent, more specific, less toxicand more effective than known inhibitors of telomerase. The designedcompounds can also be less potent but have a longer half-life in vivoand/or in vitro and therefore be more effective at modulating telomeraseactivity in vivo and/or in vitro for prolonged periods of time. Suchdesigned modulators are useful to inhibit or activate telomeraseactivity to, e.g., alter lifespan or proliferative capacity of a cell.

The present invention also provides the use of molecular designtechniques to computationally screen small molecule databases forchemical entities or compounds that can bind to telomerase in a manneranalogous to its natural substrates. Such computational screening canidentify various groups which interact with one or more amino acidresidues of a domain disclosed herein and can be employed to synthesizemodulators of the present invention.

In vitro (i.e., in solution) screening assays are also embraced by thepresent invention. For example, such assays include combiningtelomerase, the telomerase TRBD domain (e.g., as disclosed herein), orother fragments of telomerase (e.g., motifs or pockets disclosed herein)with Or without substrates or cofactors (e.g., TER, complementary DNA,nucleotides, or Mg) in solution and determining whether a test compoundcan block or enhance telomerase activity. In this respect, in vitroscreening assays can be carried out to monitor nucleotide or DNA bindingin the presence or absence of a test compound.

Compounds screened in accordance with the methods of the presentinvention are generally derived from libraries of agents or compounds.Such libraries can contain either collections of pure agents orcollections of agent mixtures. Examples of pure agents include, but arenot limited to, proteins, polypeptides, peptides, nucleic acids,oligonucleotides, carbohydrates, lipids, synthetic or semi-syntheticchemicals, and purified natural products. Examples of agent mixturesinclude, but are not limited to, extracts of prokaryotic or eukaryoticcells and tissues, as well as fermentation broths and cell or tissueculture supernates. Databases of chemical structures are also availablefrom a number of sources including Cambridge Crystallographic DataCentre (Cambridge, UK) and Chemical Abstracts Service (Columbus, Ohio).De novo design programs include Ludi (Biosym Technologies Inc., SanDiego, Calif.), Sybyl (Tripos Associates) and Aladdin (Daylight ChemicalInformation Systems, Irvine, Calif.).

Library screening can be performed using any conventional method and canbe performed in any format that allows rapid preparation and processingof multiple reactions. For in vitro screening assays, stock solutions ofthe test compounds as well as assay components can be prepared manuallyand all subsequent pipeting, diluting, mixing, washing, incubating,sample readout and data collecting carried out using commerciallyavailable robotic pipeting equipment, automated work stations, andanalytical instruments for detecting the signal generated by the assay.Examples of such detectors include, but are not limited to,luminometers, spectrophotometers, and fluorimeters, and devices thatmeasure the decay of radioisotopes.

After designing or screening for a compound which binds to at least oneamino acid residue of a domain or pocket disclosed herein, the compoundis subsequently tested for its ability to modulate the activity oftelomerase. Such activities of telomerase include telomerase catalyticactivity (which may be either processive or non-processive activity);telomerase processivity; conventional reverse transcriptase activity;nucleolytic activity; primer or substrate (telomere or synthetictelomerase substrate or primer) binding activity; dNTP binding activity;RNA (i.e., TER) binding activity; and protein binding activity (e.g.,binding to telomerase-associated proteins, telomere-binding proteins, orto a protein-telomeric DNA complex). See, e.g., assays disclosed in U.S.Pat. No. 7,262,288.

Telomerase catalytic activity is intended to encompass the ability oftelomerase to extend a DNA primer that functions as a telomerasesubstrate by adding a partial, one, or more than one repeat of asequence (e.g., TTAGGG) encoded by a template nucleic acid (e.g., TER).This activity may be processive or non-processive. Processive activityoccurs when a telomerase RNP adds multiple repeats to a primer ortelomerase before the DNA is released by the enzyme complex.Non-processive activity occurs when telomerase adds a partial, or onlyone, repeat to a primer and is then released. In vivo, however, anon-processive reaction could add multiple repeats by successive roundsof association, extension, and dissociation. This can occur in vitro aswell, but it is not typically observed in standard assays due to thevastly large molar excess of primer over telomerase in standard assayconditions. Conventional assays for determining telomerase catalyticactivity are disclosed, for example, in Morin (1989) Cell 59:521; Morin(1997) Eur. J. Cancer 33:750; U.S. Pat. No. 5,629,154; WO 97/15687; WO95/13381; Krupp, et al. (1997) Nucleic Acids Res. 25:919; Wright, et al.(1995) Nuc. Acids Res. 23:3794; Tatematsu, et al. (1996) Oncogene13:2265.

Telomerase conventional reverse transcriptase activity is described in,e.g., Morin (1997) supra, and Spence, et al. (1995) Science 267:988.Because telomerase contains conserved amino acid motifs that arerequired for reverse transcriptase catalytic activity, telomerase hasthe ability to transcribe certain exogenous (e.g., non-TER) RNAs. Aconventional RT assay measures the ability of the enzyme to transcribean RNA template by extending an annealed DNA primer. Reversetranscriptase activity can be measured in numerous ways known in theart, for example, by monitoring the size increase of a labeled nucleicacid primer (e.g., RNA or DNA), or incorporation of a labeled dNTP. See,e.g., Ausubel, et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y.

Because telomerase specifically associates with TER, the DNA primer/RNAtemplate for a conventional RT assay can be modified to havecharacteristics related to TER and/or a telomeric DNA primer. Forexample, the RNA can have the sequence (CCCTAA)_(n), where n is at least1, or at least 3, or at least 10 or more. In one embodiment, the(CCCTAA)_(n) region is at or near the 5′ terminus of the RNA (similar tothe 5′ locations of template regions in telomerase RNAs). Similarly, theDNA primer may have a 3′ terminus that contains portions of the TTAGGGtelomere sequence, for example X_(n)TTAG, X_(n)AGGG, etc., where X is anon-telomeric sequence and n is 6-30. In another embodiment, the DNAprimer has a 5′ terminus that is non-complementary to the RNA template,such that when the primer is annealed to the RNA, the 5′ terminus of theprimer remains unbound. Additional modifications of standard reversetranscription assays that may be applied to the methods of the inventionare known in the art.

Telomerase nucleolytic activity is described in, e.g., Morin (1997)supra and Collins & Grieder (1993) Genes Dev. 7:1364. Telomerasepreferentially removes nucleotides, usually only one, from the 3′ end ofan oligonucleotide when the 3′ end of the DNA is positioned at the 5′boundary of the DNA template sequence, in humans and Tetrahymena, thisnucleotide is the first G of the telomeric repeat (TTAGG in humans).Telomerase preferentially removes G residues but has nucleolyticactivity against other nucleotides. This activity can be monitored usingconventional methods known in the art.

Telomerase primer (telomere) binding activity is described in, e.g.,Morin (1997) supra; Collins, et al. (1995) Cell 81:677; Harrington, etal. (1995) J. Biol. Chem. 270:8893. There are several ways of assayingprimer binding activity; however, a step common to most methods isincubation of a labeled DNA primer with telomerase or telomerase/TERunder appropriate binding conditions. Also, most methods employ a meansof separating unbound DNA from protein-bound DNA. Such methods caninclude, e.g., gel-shift assays or matrix binding assays. The DNA primercan be any DNA with an affinity for telomerase, such as, for example, atelomeric DNA primer like (TTAGGG)_(n), where n could be 1-10 and istypically 3-5. The 3′ and 5′ termini can end in any location of therepeat sequence. The primer can also have 5′ or 3′ extensions ofnon-telomeric DNA that could facilitate labeling or detection. Theprimer can also be derivatized, e.g., to facilitate detection orisolation.

Telomerase dNTP binding activity is described in, e.g., Morin (1997)supra and Spence, et al. (1995) supra. Telomerase requires dNTPs tosynthesize DNA. The telomerase protein has a nucleotide binding activityand can be assayed for dNTP binding in a manner similar to othernucleotide binding proteins (Kantrowitz, et al. (1980) Trends Biochem.Sci. 5:124). Typically, binding of a labeled dNTP or dNTP analog can bemonitored as is known in the art for non-telomerase RT proteins.

Telomerase RNA (i.e., TER) binding activity is described in, e.g., Morin(1997) supra; Harrington, et al. (1997) Science 275:973; Collins, et al.(1995) Cell 81:677. The RNA binding activity of a telomerase protein ofthe invention may be assayed in a manner similar to the DNA primerbinding assay described supra, using a labeled RNA probe. Methods forseparating bound and unbound RNA and for detecting RNA are well known inthe art and can be applied to the activity assays of the invention in amanner similar to that described for the DNA primer binding assay. TheRNA can be full length TER, fragments of TER or other RNAs demonstratedto have an affinity for telomerase or TRBD. See U.S. Pat. No. 5,583,016and WO 96/40868.

In addition to determining either RNA or DNA binding, assays monitoringbinding of the RNA-templating region and the complementary telomeric DNAsequence can also be carried out. By way of illustration, Example 5describes the use of fluorescence polarization to determine binding ofcompounds to TERT protein.

To further evaluate the efficacy of a compound identified using themethod of the invention, one of skill will appreciate that a modelsystem of any particular disease or disorder involving telomerase can beutilized to evaluate the adsorption, distribution, metabolism andexcretion of a compound as well as its potential toxicity in acute,sub-chronic and chronic studies. For example, the effector or modulatorycompound can be tested in an assay for replicative lifespan inSaccharomyces cerevisiae (Jarolim, et al. (2004) FEMS Yeast Res.5(2):169-77). See also, McChesney, et al. (2005) Zebrafish 1(4):349-355and Nasir, et al. (2001) Neoplasia 3(4):351-359, which describe marinemammal and dog tissue model systems for analyzing telomerase activity.

As described in the Examples and listed in the Tables herein, a numberof effector molecules were identified using the instant screeningassays. It was found that telomerase effector molecules fell intoparticular classes including benzohydrazides, benzoic acids,carboxamides, carboxylic acids, acrylamides, isophthalic acids,1,3-dihydro-2H-indol-2-ones, acetamides, benzamides,naphthalenedisulfonates, 1,3-dioxo-5-isoindolinecarboxamides,dicarbonimidic diamides, ethanediamides, piperidinecarboxamides, and2-quinazolinyl guanidines. Therefore, compounds of these classes are ofparticular use in modulating the activity of telomerase. Morespecifically, compounds of Formula I-IV are of use in methods ofinhibiting the activity of telomerase and in the treatment of diseasesor conditions as described herein.

Compounds that bind to at least one amino acid residue of one or more ofthe telomerase domains or pockets disclosed herein can be used in amethod for modulating (i.e., blocking or inhibiting, or enhancing oractivating) a telomerase. Such a method involves contacting a telomeraseeither in vitro or in vivo with an effective amount of a compound thatinteracts with at least one amino acid residue of a domain or pocket ofthe invention so that the activity of telomerase is modulated. Aneffective amount of an effector or modulatory compound is an amountwhich reduces or increases the activity of the telomerase by at least30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to telomerasenot contacted with the compound. Such activity can be monitored byenzymatic assays detecting activity of the telomerase or by monitoringthe expression or activity of proteins which are known to be associatedwith or regulated by telomerase.

One of skill in the art can appreciate that modulating the activity oftelomerase can be useful in selectively analyzing telomerase signalingevents in model systems as well as in preventing or treating diseases,conditions, and disorders involving telomerase. The selection of thecompound for use in preventing or treating a particular disease ordisorder will be dependent upon the particular disease or disorder. Forexample, human telomerase is involved in cancer and therefore a compoundwhich inhibits telomerase will be useful in the prevention or treatmentof cancer including solid tumors (e.g., adenocarcinoma of the breast,prostate, and colon; melanoma; non-small cell lung; glioma; as well asbone, breast, digestive system, colorectal, liver, pancreatic,pituitary, testicular, orbital, head and neck, central nervous system,acoustic, pelvic, respiratory tract, and urogenital neoplasms) andleukemias (e.g., B-cell, mixed-cell, null-cell, T-cell, T-cell chronic,lymphocytic acute, lymphocytic chronic, mast-cell, and myeloid). Cancercells (e.g., malignant tumor cells) that express telomerase activity(telomerase-positive cells) can be mortalized by decreasing orinhibiting the endogenous telomerase activity. Moreover, becausetelomerase levels correlate with disease characteristics such asmetastatic potential (e.g., U.S. Pat. Nos. 5,639,613; 5,648,215;5,489,508; Pandita, et al. (1996) Proc. Am. Ass. Cancer Res. 37:559),any reduction in telomerase activity could reduce the aggressive natureof a cancer to a more manageable disease state (increasing the efficacyof traditional interventions).

By way of illustration, Example 9 describes cell-based assays and animalmodel systems which are useful for assessing the inhibition of tumorcell growth by one or more compounds of the invention. Another usefulmethod for assessing anticancer activities of compounds of the inventioninvolves the multiple-human cancer cell line screening assays run by theNational Cancer Institute (see, e.g., Boyd (1989) in Cancer: Principlesand Practice of Oncology Updates, DeVita et al., eds, pp. 1-12). Thisscreening panel, which contains approximately 60 different human cancercell lines, is a useful indicator of in vivo antitumor activity for abroad variety of tumor types (Greyer, et al. (1992) Seminars Oncol.19:622; Monks, et al. (1991) Natl. Cancer Inst. 83:757-766), such asleukemia, non-small cell lung, colon, melanoma, ovarian, renal,prostate, and breast cancers. Antitumor activities can be expressed interms of ED₅₀ (or GI₅₀), where ED₅₀ is the molar concentration ofcompound effective to reduce cell growth by 50%. Compounds with lowerED₅₀ values tend to have greater anticancer activities than compoundswith higher ED₅₀ values.

Upon the confirmation of a compound's potential activity in one or morein vitro assays, further evaluation is typically conducted in vivo inlaboratory animals, for example, measuring reduction of lung nodulemetastases in mice with B16 melanoma (e.g., Schuchter, et al. (1991)Cancer Res. 51:682-687). The efficacy of a compound of the inventioneither alone or as a drug combination chemotherapy can also beevaluated, for example, using the human B-CLL xenograft model in mice(e.g., Mohammad, et al. (1996) Leukemia 10:130-137). Such assaystypically involve injecting primary tumor cells or a tumor cell lineinto immune compromised mice (e.g., a SCID mouse or other suitableanimal) and allowing the tumor to grow. Mice carrying the tumors arethen treated with the compound of interest and tumor size is measured tofollow the effect of the treatment. Alternatively, the compound ofinterest is administered prior to injection of tumor cells to evaluatetumor prevention. Ultimately, the safety and efficacy of compounds ofthe invention are evaluated in human clinical trials.

Compounds that activate or stimulate telomerase activity can be used inmethods for treating or preventing a disease or condition induced orexacerbated by cellular senescence in a subject; methods for decreasingthe rate of senescence of a subject, e.g., after onset of senescence;methods for extending the lifespan of a subject; methods for treating orpreventing a disease or condition relating to lifespan; methods fortreating or preventing a disease or condition relating to theproliferative capacity of cells; and methods for treating or preventinga disease or condition resulting from cell damage or death. Certaindiseases of aging are characterized by cell senescence-associatedchanges due to reduced telomere length (compared to younger cells),resulting from the absence (or much lower levels) of telomerase activityin the cell. Telomerase activity and telomere length can be increasedby, for example, increasing the activity of telomerase in the cell. Apartial listing of conditions associated with cellular senescence inwhich increased telomerase activity can be therapeutic includesAlzheimer's disease, Parkinson's disease, Huntington's disease, andstroke; age-related diseases of the integument such as dermal atrophy,elastolysis and skin wrinkling, graying of hair and hair loss, chronicskin ulcers, and age-related impairment of wound healing; degenerativejoint disease; osteoporosis; age-related immune system impairment (e.g.,involving cells such as B and T lymphocytes, monocytes, neutrophils,eosinophils, basophils, NK cells and their respective progenitors);age-related diseases of the vascular system; diabetes; and age-relatedmacular degeneration. Moreover, telomerase activators can be used toincrease the proliferative capacity of a cell or in cellimmortalization, e.g., to produce new cell lines (e.g., most humansomatic cells).

Prevention or treatment typically involves administering to a subject inneed of treatment a pharmaceutical composition containing an effectiveof a compound identified via screening methods of the invention. In mostcases this will be a human being, but treatment of agricultural animals,e.g., livestock and poultry, and companion animals, e.g., dogs, cats andhorses, is expressly covered herein. The selection of the dosage oreffective amount of a compound is that which has the desired outcome ofpreventing, reducing or reversing at least one sign or symptom of thedisease or disorder being treated. Methods for treating cancer and othertelomerase-related diseases in humans are described in U.S. Pat. Nos.5,489,508, 5,639,613, and 5,645,986. By way of illustration, a subjectwith cancer (including, e.g., carcinomas, melanomas, sarcomas, lymphomasand leukaemias) can experience unexplained weight loss, fatigue, fever,pain, skin changes, sores that do not heal, thickening or lump in breastor other parts of the body, or a nagging cough or hoarseness, whereintreatment with a compound of the invention can prevent, reduce, orreverse one or more of these symptoms.

Pharmaceutical compositions of the present invention include compoundsof the invention, or pharmaceutically acceptable salts or complexesthereof, in admixture with a a pharmaceutically acceptable carrier. Suchpharmaceutical compositions can be prepared by methods and containcarriers which are well-known in the art. A generally recognizedcompendium of such methods and ingredients is Remington: The Science andPractice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. LippincottWilliams & Wilkins: Philadelphia, Pa., 2000. Apharmaceutically-acceptable carrier, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, is involved in carrying or transporting the subject compoundfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be acceptable in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject being treated.

Examples of materials which can serve as pharmaceutically acceptablecarriers include sugars, such as lactose, glucose and sucrose; starches,such as corn starch and potato starch; cellulose, and its derivatives,such as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients, such ascocoa butter and suppository waxes; oils, such as peanut oil, cottonseedoil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;glycols, such as propylene glycol; polyols, such as glycerin, sorbitol,mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyllaurate; agar; buffering agents, such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides; and other non-toxic compatiblesubstances employed in pharmaceutical formulations. Wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

Compounds of the present invention can be used alone or in combinationwith other agents, such as cancer chemotherapeutic agents, in thetreatment of disease. Thus, in particular embodiments, the presentinvention embraces combining an effective amount of a compound of theinvention with one or more chemotherapeutic agents or antiproliferativeagents. The drug combination can be included in the same or multiplepharmaceutical compositions. In addition, the individual drugs can beadministered simultaneously or consecutively (e.g., immediatelyfollowing or within an hour, day, or month of each other).

More particularly, cancer chemotherapeutic agents refer to agents thatinduce apoptosis and/or impair mitosis of rapidly dividing cells. Cancerchemotherapeutic agents of use in accordance with the present inventioninclude, but are not limited to, alkylating agents (e.g., cisplatin,carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, andchlorambucil); antimetabolites (e.g., azathioprine and mercaptopurine);anthracyclines; plant products including vinca alkaloids (e.g.,Vincristine, Vinblastine, Vinorelbine, and Vindesine) and taxanes (e.g.,paclitaxel); and topoisomerase inhibitors (e.g., amsacrine, irinotecan,topotecan, etoposide, etoposide phosphate, and teniposide), which affectcell division or DNA synthesis and/or function; as well as monoclonalantibodies (e.g., trastuzumab, cetuximab, rituximab, and Bevacizumab)and tyrosine kinase inhibitors such as imatinib mesylate, which directlytarget a molecular abnormality in certain types of cancer (e.g., chronicmyelogenous leukemia, gastrointestinal stromal tumors).

A radiotherapeutic agent refers to an agent the produces ionizingradiation that damages cellular DNA. Radiotherapy is conventionallyprovided as external beam radiotherapy (EBRT or XBRT) or teletherapy,brachytherapy or sealed source radiotherapy, and systemic radioisotopetherapy or unsealed source radiotherapy. The differences relate to theposition of the radiation source; external is outside the body,brachytherapy uses sealed radioactive sources placed precisely in thearea under treatment, and systemic radioisotopes are given by infusionor oral ingestion. In this regard, when used in the context of acomposition of the present invention, a radiotherapeutic is intended tomain a radioactive agent used in brachytherapy. When used in the contextof the methods of the present invention, a cancer therapeutic includesall forms of radiotherapy routinely used in the art.

The selection of one or more appropriate cancer therapeutics for use inthe composition and methods of the invention can be carried out theskilled practitioner based upon various factors including the conditionof the patient, the mode of administration, and the type of cancer beingtreated. Moreover, the combination therapy can be included in the sameor multiple pharmaceutical compositions. In addition, the individualdrugs can be administered simultaneously or consecutively (e.g.,immediately following or within an hour, day, or month of each other).

The compositions of the present invention can be administeredparenterally (for example, by intravenous, intraperitoneal, subcutaneousor intramuscular injection), topically (including buccal andsublingual), orally, intranasally, intravaginally, or rectally accordingto standard medical practices.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of a compound at levels lower than that required in order toachieve the desired therapeutic effect and gradually increase the dosageuntil the desired effect is achieved. This is considered to be withinthe skill of the artisan and one can review the existing literature on aspecific compound or similar compounds to determine optimal dosing.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Structure of Tetrahymena thermophila TERT

Protein Expression and Purification.

The T. thermophila TERT residues 254-519 was identified by limitedproteolysis and cloned into a modified version of the pET28b vectorcontaining a cleavable hexa-histidine tag at its N-terminus. The proteinwas over-expressed in E. coli BL21 (pLysS) at 20° C. for 4 hours. Thecells were lysed by sonication in 50 mM Tris-HCl, 10% glycerol, 0.5 MKCl, 5 mM β-mercaptoethanol, and 1 mM PMSF, pH 7.5 on ice. The proteinwas first purified over a Ni-NTA column followed by TEV cleavage of thehexa-histidine tag overnight at 4° C. The TRBD/TEV mix was diluted sothat the concentration of imidazole was at 15 mM and the protein mix waspassed over a Ni-NTA column to remove the TEV, the cleaved tag and anytagged protein. The Ni-NTA flow through was concentrated to 1 ml anddiluted to a salt concentration of 0.15 M. The diluted TRBD sample wasthen passed over a POROS-HS column (PerSeptive Biosystems, Framingham,Mass.). At this stage, the protein was more than 99% pure. The proteinwas finally passed over a SEPHADEX-S200 sizing column pre-equilibratedwith 50 mM Tris-HCl, 10% glycerol, 0.5 M KCl, and 2 mM DTT, pH 7.5 toremove any TRBD aggregates. The pure, monodisperse protein as indicatedby SDS-page and dynamic light scattering, respectively, was concentratedto 8 mg/ml using an AMICON 10K cutoff (MILLIPORE, Billerica, Mass.) andthe protein was stored at 4° C. for subsequent studies. Stock proteinwas dialyzed in 5 mM Tris-HCl, 500 mM KCl, 1 mM TCEP, pH 7.5 prior tocrystallization trials.

Protein Crystallization and Data Collection.

Initial plate-like clusters of TRBD that diffracted poorly (˜4 Åresolution) were grown at 4° C. using the sitting drop method by mixingon volume of dialyzed protein with one volume of reservoir solutioncontaining 20% PEG 3350, 0.2 M NaNO₃. Single, well diffracting crystalswere grown in microbatch trays under paraffin oil by mixing one volumeof dialyzed protein with an equivalent volume of 50 mM HEPES (pH 7.0),44% PEG 400, 0.4 M NaNO₃, 0.4 M NaBr and 1 mM TCEP at 4° C. Crystalswere harvested into cryoprotectant solution that contained 25 mM HEPES(pH 7.0), 25% PEG 400, 0.2 M NaNO₃, 0.2 M NaBr and 1 mM TCEP and wereflash frozen in liquid nitrogen. Data were collected at the NSLS, beamline X6A and processed with HKL-2000 (Minor (1997) Meth. Enzymol.Macromole. Crystallogr. Part A 276:307-326) (Table 3). The crystalsbelong to the monoclinic space group P2₁ with one monomer in theasymmetric unit.

TABLE 3 Native Holmium-Derivative TRBD₍₂₅₄₋₅₁₉₎ λ Ho-λ1 Ho-λ2 Wavelength0.9795 1.5347 1.5595 (Å) Space group P2₁ P2₁ P2₁ Cell 39.4   67.239.2   68.2 39.2   68.2 dimensions (Å) 51.5 90.7 50.1 91.6 50.1 91.6Resolution (Å)  20-1.71   50-2.59   50-2.63 (1.77-1.71)* (2.69-2.59)(3.02-2.63) Redundancy 3.7 (3.0) 1.7 (1.8) 1.7 (1.8) Completeness 99.3(93.3) 92.5 (88.1) 92.9 (88.7) (%) R_(sym) (%)  4.7 (48.1)  7.3 (23.8) 7.0 (21.5) I/σ (I) 27.3 (2.6)    9 (3.4) 9.4 (3.7) Phasing AnalysisResolution (Å) 50-2.7 Number of sites 2 Mean figure of merit (FOM) 0.43*Values in parentheses correspond to the highest resolution shell.

Structure Determination and Refinement.

Initial phases were obtained from a two-wavelength MAD holmium (Ho)derivative that was prepared by co-crystallizing the protein with 5 mMHoCl₃. Heavy atom sites were located using SOLVE (Terwilliger (2003)Methods Enzymol. 374:22-37) and the sites were refined and new phasescalculated with MLPHARE (CCP4 (1994) Acta Crystallogr. D 50:760-763) asimplemented in ELVES (Holton & Alber (2004) Proc. Natl. Acad. Sci. USA101:1537-1542) (Table 3). Initial maps showed well-defined density onlyfor the larger half of the molecule. The density for the smaller half ofthe molecule was weak mostly due to its intrinsic mobility with respectto larger half of the molecule. The problem associated with building themodel into the density was exacerbated by the lack of informationregarding the location of specific side chains such asselenomethionines. Key factors in building a complete model weresuccessive rounds of PRIME and SWITCH in RESOLVE (Terwilliger (2002)Acta Crystallogr. D Biol. Crystallogr. 58:1937-1940) followed by manualbuilding in COOT (Emsley & Cowtan (2004) Acta Crystallogr. D Biol.Crystallogr. 60:2126-2132). The model was refined using both CNS-SOLVE(Brunger, et al. (1998) Acta Crystallogr. D Biol. Crystallogr.54:905-921) and REFMAC5 (Murshudov, et al. (1997) Acta Crystallogr. DBiol. Crystallogr. 53:240-255). The last cycles of refinement werecarried out with TLS restraints as implemented in REFMAC5 (Table 4).Figures were prepared in PYMOL (DeLano (2002)) and electrostaticsurfaces in APBS (Baker, et al. (2001) Proc. Natl. Acad. Sci. USA98:10037-10041).

TABLE 4 TRBD₍₂₅₄₋₅₁₉₎ Refinement Statistics Resolution (Å) 20-1.71R_(work)/R_(free) (%) 20.0/23.9 RMSD bonds (Å) 0.008 RMSD angles (°)0.831 Number of atoms Protein 2145 Bromine 7 Water 213 Average B (Å²)Protein 27.41 Bromine 42.63 Water 31.22 Ramachandran % (no res.) Mostfavored 91.6 Allowed 8.4

TRBD Structure.

To explore the role of the essential RNA-binding domain of telomerase(TRBD), a construct identified by limited proteolysis, containingresidues 254-519 from T. thermophila (FIG. 1A) was purified tohomogeneity. This protein construct was monomeric in solution asindicated by both gel filtration and dynamic light scattering. Crystalsof this construct grew readily and diffracted to 1.71 Å resolution(Table 4). The protein was phased to 2.7 Å resolution by themultiwavelength anomalous dispersion method (MAD) using a holmiumderivative and the phases were extended with the native dataset to 1.71Å resolution (Table 4). In the refined structure there was clear densityfor residues 257-266 and 277-519.

The structure contains twelve α-helices linked together by several longloops and two short β-strands. The helices are organized so that themolecule is divided into two asymmetric halves linked together by threeextended loops. The larger half is composed of nine α-helices, one ofwhich (α11) runs along the middle of the domain and spans its entirelength making contacts with all other eight helices. The smaller half ofthe molecule is composed of three helices (α4, α5 and α12), all of whichare arranged at a ˜120° angle to the plane of the larger half of theprotein. The smaller half of the protein is somewhat more flexible thanthe larger half as suggested by its high B factors reflecting theintrinsic mobility of this region and may result from the absence ofobservable contacts with the RNA substrate. An interesting feature ofthe structure is a β-hairpin formed by the 15-residues that connecthelices all and α12 of the larger and the smaller halves, respectively.The β-hairpin protrudes from the base of the crevice formed by the twohalves of the protein and stands at a 45° angle to the plane of thesmaller half of the molecule. The positioning and the fact that thishairpin is well-defined in the density could be attributed to helix α7and the loop that connects it to helix α8. Both of these elements areconveniently positioned at the back of this hairpin holding it in place.A search in the protein structure database using the Dali server (Holm &Sander (1996) Science 273:595-603) produced no structural homologues,indicating that the TRBD domain of telomerase is a novel nucleic acidbinding fold. The overall organization of the two halves of the proteinhas significant implications for nucleic acid recognition and binding.

The TRBD RNA-Binding Motifs.

The ability of the TRBD domain to interact with TER has been attributedto two conserved motifs known as the CP-, and T-motifs, while a thirdmotif known as the QFP-motif is thought to be important for RNP assembly(FIGS. 2A and 2B) (Bosoy, et al. (2003) J. Biol. Chem. 278:3882-3890;Bryan, et al. (2000) supra; Jacobs, et al. (2005) supra; Xia, et al.(2000) Mol. Cell. Biol. 20:5196-5207). The TRBD structure shows that theQFP-motif is formed by several mostly hydrophobic residues, which arelocated on the larger half of the molecule and are buried within thecore of the domain making extensive hydrophobic contacts with thesurrounding residues aiding in the fold of the protein. These residuesincluded Gln375, Ile376, Leu380, Ile383, Ile384, Cys387, Val388, Pro389,Leu392, Leu393, Asn397, Leu405, Phe408, Tyr422, Ile423, Met426, Trp433,and Phe434. The location and the contacts of the QFP-residues indicatethat they are not directly involved in nucleic acid binding.

The T-motif is located at the center of the molecule where the twohalves of the protein meet and it is composed of residues that form bothpart of the β-hairpin and helix α12. Together these structural elementsform a narrow (˜10 Å), well-defined pocket (T-pocket) that is lined byseveral solvent exposed and highly conserved residues (Phe476, Tyr477,Thr479, Glu480, Tyr491, Arg492, Lys493, and Trp496). Of particular noteare the side chains of the invariant residues Tyr477 and Trp496, whichare part of the β-hairpin and helix α12, respectively. Together theseresidues form a “hydrophobic pincer” that could sandwich thepurine/pirimidine moiety of an interacting RNA nucleotide. In thisstructure, the side chains of Tyr477 and Trp496 are only 4 Å apart,which is not sufficient to accommodate a nucleotide base. Insertion of abase between the two side chains would require structural rearrangementof the T-pocket, possibly splaying of the two halves of the moleculesapart. In addition to its hydrophobic part, the T-pocket also containsseveral hydrophilic residues such as Arg492 and Lys493 both of which aresolvent exposed and are located at the interface of the T- and CP-pocketconnecting the two together.

The CP-motif is formed by helix α3 and the following loop. In contrastto the T-motif, which is a narrow well-defined pocket, the CP-motif iscomposed a shallow, wide (˜20 Å), highly positively charged cavitylocated adjacent and beneath the entry of the T-pocket. Several of theconserved residues that form the CP-motif include Phe323, Leu327,Lys328, Lys329, Cys331, Leu333, and Pro334. These residues are buried inthe core of the larger half or the region that connects the two halvesof the molecule and are contributing to the protein fold. Of particularinterest are residues Leu327, Cys331, Leu333 and Pro334 all of which areburied and make direct contacts with structural elements of the T-motifthus aiding in the formation of both the CP- and the T-pockets. Forexample, Leu327 and Cys331 are within Van der Waal contacts of the largehydrophobic side chain of the invariant Phe476 and the aliphatic part ofthe side chain of the conserved Arg492 both of which form part of theβ-hairpin. Interestingly, Arg492 is located at the base of helix α12 andits contact with Leu327, Cys331, and Leu333 partially helps positionthis helix at a 45° angle of the plane that runs parallel with thelarger half of the molecule thus further facilitating the formation ofthe T-pocket. Moreover, the interaction of Arg492 with Leu327, Cys331,and Leu333 helps position the guanidine moiety, the only solvent-exposedpart of this residue, at the interface formed by the CP- and T-pockets.The CP-pocket also contains several surface-exposed, conserved residuesthat are mainly hydrophilic in nature. These include Lys328 and Lys329both of which are located beneath the T-pocket and in close proximity ofArg492 and Lys493 together forming a single large, positively chargedsurface area that almost spans the entire side of the molecule.

TRBD Structure and Existing Mutants.

Several mutants of TERT that affect RNA-binding and telomerase activityhave been isolated. Several of these mutants are found in the TRBDdomain and specifically within the T- and CP-motifs. Single- and double-as well as stretches of 4-10 amino acid alanine substitutions withinthese two motifs showed moderate to severe loss (20-100%) of RNA-bindingaffinity and polymerase activity when compared to the wild type enzyme(Bryan, et al. (2000) supra; Lai, et al. (2002) supra; Miller, et al.(2000) supra).

One set of mutants, Phe476Ala, Tyr477Ala, Thr479Ala, Glu480Ala,Arg492Ala and Trp496Ala, showed severe loss (80-100%) of RNA-bindingaffinity and telomerase activity suggesting that these residues mediatedirect contacts with the RNA substrate (Bryan, et al. (2000) supra; Lai,et al. (2002) supra). All five residues are part of the T-motif and,with the exception of Phe476, all of their side chains are solventexposed. In the structure, both Tyr477 and Trp496 are located at thebase of the T-pocket and their side chains form a “hydrophobic pincer”.Assuming that the solvent-exposed side chains of these residues areinvolved in stacking interactions with the ssRNA, mutating them to smallalanines would likely compromise substrate binding which explains thedramatic loss of RNA-binding affinity and telomerase function. Incontrast to Tyr477 and Trp496, Phe476 is buried and is not accessiblefor interactions with the nucleic acid substrate. Instead, Phe476 islocated at the base of the β-hairpin and contributes significantly tothe formation of the T-pocket. Mutating the large hydrophobic side chainof this residue to the small alanine would likely lead to conformationalrearrangements of this pocket and loss of RNA-binding affinity andtelomerase activity.

A second set of alanine mutants, Leu327Ala, Lys329Ala, Cys331Ala, andPro334Ala, which showed moderate loss of RNA-binding affinity andtelomerase activity has also been isolated (Bryan, et al. (2000) supra;Miller, et al. (2000) supra). Both Leu327 and Cys331 make directcontacts with Phe476 and the aliphatic part of the side chain of Arg492,both of which are located at the base of the T-motif. Mutation to thesmaller alanine residue could result in the rearrangement of theT-pocket potentially leading to loss of interactions with the nucleicacid substrate and loss of function. Likewise, Pro334 is located at theback of helix α12 and makes direct contacts with residues of thisstructural element. Helix α12 contains the invariant Trp496 and theconserved Lys493, both of which form part of the T-pocket. MutatingPro334 into an alanine could lead to the displacement of helix α10 andreorganization of the T-pocket leading to loss of function. Lys329 isalso located on helix α3 and unlike Leu327Ala, Cys331Ala, and Pro334Ala,is solvent exposed possibly making direct contacts with the nucleic acidsubstrate. Mutating it to an alanine would lead to lose of RNAinteractions and loss of RNA-binding affinity and telomerase activity.

TRBD Domain-Mediated Formation of Stable RNP Complex and Repeat AdditionProcessivity.

In vivo, telomerase exists as a stable ribonucleoprotein complex andcontacts between the protein (TERT) and the RNA components (TER) aremediated by the TEN, TRBD and the RT domains. Extensive biochemical andmutagenesis studies have shown that the TRBD is involved in extensive,specific interactions with stem I and the TBE of TER (Lai, et al. (2001)supra; O'Connor, et al. (2005) supra) (FIG. 4). Contacts between theTRBD and TER are thought to facilitate the proper assembly andstabilization of the RNP complex and promote repeat additionprocessivity (Lai, et al. (2003) supra). In ciliates, in addition to theTRBD, a conserved motif (CP2) located N-terminally to the TRBD domain isthought to be required for TERT-TER assembly and template boundarydefinition (Lai, et al. (2002) supra; Miller, et al. (2000) supra).However, until now it has been unclear as to how the telomerase TRBDcarries out this process. The present analysis indicates that the TRBDdomain is divided into two asymmetric halves connected by several longloops that are shaped like a boomerang, an arrangement that hassignificant implications for RNA recognition and binding. The overallorganization of the two lobes of the molecule results in the formationof two well-defined cavities (CP- and T-pockets) on the surface of theprotein that consist of several solvent-exposed, invariant/conservedresidues. The T-pocket is a narrow, deep cavity located at the junctionof the two halves of the molecule with part of it being hydrophobic innature while the part that is located in proximity of the CP-pocket ispositively charged. Interestingly, the hydrophobic side chains of Tyr477and Trp496 are solvent-exposed and are stacked against each otherforming a narrow “hydrophobic pincer” that in this structure could notaccommodate a nucleotide base. It is, however, worth noting that helixα12, which contains Trp496, is somewhat flexible with respect to theβ-hairpin that contains Tyr477. The ability of helix α12 and thereforeTrp496 to move could splay the two side chains apart thus allowing forthe space required for the accommodation of a nucleotide base betweenthem. Another possibility is that the polar moiety of Tyr477 and Trp496could act together as a nucleotide base that would allow for theformation of pseudo Watson Crick interactions with an incomingnucleotide base. Pseudo Watson Crick interactions have been previouslyobserved for a number of protein nucleic acid complexes including theRho transcription termination factor (Bogden, et al. (1999) Mol. Cell.3:487-493) and the signal recognition particle (Wild, et al. (2001)Science 294:598-601). The width and the organization of the hydrophobicpart of the T-pocket indicate that it binds ssRNA, most likely the TBE,possibly mediated by a network of stacking interactions.

In contrast to the T-pocket, the CP-pocket is a positively charged,shallow cavity located on the side of the molecule and forms anextension of the T-pocket. Together the hydrophilic part of the T-pocketand the CP-pocket are lined with several lysines and arginines the sidechains of which are solvent exposed and could be involved in directcontacts with the backbone of double-stranded RNA. The width and thechemical nature of this pocket indicate that it binds double-strandedRNA, most likely stem I or stem II (FIG. 4). The nature and the extentof the protein/nucleic acid interactions mediated by the TRBD bindingpockets provides the stability required for the proper assembly of afunctional ribonucleoprotein enzyme and guide TERT to a TER binding site(between stem I and II) that has significant implications for telomerasefunction.

Telomerase is unique in its ability to add multiple shortoligonucleotide repeats at the end of linear chromosomes. The enzyme'sability to do so has been partly attributed to the interactions of theTRBD domain with the TBE and in ciliates both the TRBD and the CP2 motif(Lai, et al. (2002) supra; Lai, et al. (2003) supra; Miller, et al.(2000) supra). The TBE is composed of stem II and the flanking ssRNAregions and is located downstream of stem I and only a few nucleotidesupstream of the RNA template (FIG. 4). The TRBD structure indicates thatthe T-pocket, a narrow, hydrophobic cavity located on the surface of theprotein that can only accommodate ssRNA, may play an important role inthis process. Assuming that the T-pocket binds the ssRNA that connectsstem I and stem II, this interaction likely forces stem II to act as asteric block, which in turn forces the TRBD domain to stay within theboundaries of stem I and stem II. The stem I- and II-locked TRBD domainthen may act as an anchor that constrains the distance the RT domain cantravel and prevents it from moving beyond the boundaries of the RNAtemplate thus promoting telomerase addition processivity. In ciliateshowever, the TRBD domain alone is not sufficient for template boundarydefinition and it requires the action of the CP2 motif (Lai, et al.(2002) supra; Miller, et al. (2000) supra). It is contemplated that CP2binding to TER promotes template boundary definition either viacontributing to the stabilization of the RNP complex or, like the TRBD,it may act as an anchor that prevents slippage of the active site of theRT domain beyond the RNA template.

Example 2 Structure of Tribolium castaneum TERT in Complex with DNASubstrate

Protein Expression and Purification.

The synthetic gene of T. castaneum full-length TERT was cloned into amodified version of the pET28b vector containing a cleavablehexahistidine tag at its N-terminus. The protein was over-expressed inE. coli BL21 (pLysS) at 30° C. for 4 hours. The cells were lysed bysonication in 50 mM Tris-HCl, 10% glycerol, 0.5 M KCl, 5 mMβ-mercaproethanol, and 1 mM PMSF, pH 7.5 on ice. The protein was firstpurified over a Ni-NTA column followed by TEV cleavage of thehexahistidine tag overnight at 4° C. The TERT/TEV mixture was dialyzedto remove the excess imidazole and the protein was further purified overa second Ni-NTA column that was used to remove all his-tagged products.The Ni-NTA flow through was then passed over a POROS-HS column(Perseptive Biosystems) to remove any trace amounts of proteincontaminants. At this stage the protein was more than 99% pure. Theprotein was finally purified over a SEPHEDEX-S200 sizing columnpre-equilibrated with 50 mM Tris-HCl, 10% glycerol, 0.5 M KCl, and 1 mMTris(2-Carboxyethyl)phosphine (TCEP), pH 7.5 to remove any TERTaggregates and the protein was concentrated to 10 mg/ml using an AMICON30K cutoff (MILLIPORE) and stored at 4° C. for subsequent studies. Stockprotein was dialyzed in 10 mM Tris-HCl, 200 mM KCl, 1 mM TCEP, pH 7.5prior to crystallization trials.

Protein Crystallization and Data Collection.

Initial crystal trials of the protein alone did not produce crystals.Co-crystallization of the protein with single stranded telomeric DNA((TCAGG)₃) produced two rod-like crystal forms one of which belongs tothe orthorhombic space group P2₁2₁2₁ and diffracted to 2.71 Å and theother to the hexagonal space group P6₁ and diffracted to 3.25 Åresolution. The protein nucleic acid mix was prepared prior to settingcrystal trials by mixing one volume of dialyzed protein with 1.2-foldexcess of the DNA substrate. Both crystal forms where grown by the vapordiffusion, sitting drop method by mixing on volume of the protein-DNAmix with one volume of reservoir solution. Orthorhombic crystals wheregrown in the presence of 50 mM HEPES, (pH 7.0) and 1.5 M NaNO₃ whilehexagonal crystals grew in the presence of 100 mM Tris (pH 8.0) and 2 M(NH₄)₂SO₄ and both at room temperature. Orthorhombic crystals wereharvested into cryoprotectant solution that contained 50 mM HEPES (pH7.0), 25% glycerol, 1.7 M NaNO₃, 0.2 M KCl and 1 mM TCEP and were flashfrozen in liquid nitrogen. Hexagonal crystals were harvested intocryoprotectant solution that contained 100 mM Tris (pH 8.0), 25%glycerol, 2 M (NH₄)₂SO₄, 0.2 M KCl and 1 mM TCEP and were also flashfrozen in liquid nitrogen. Data were collected at the NSLS, beam lineX6A and processed with HKL-2000 (Minor (1997) Methods in Enzymology:Macromolecular Crystallography, part A 276:307-326) (Table 5). Bothcrystal forms contain a dimer in the asymmetric unit.

TABLE 5 Native 1 Native 2 Hg1 Hg2 Data collection Space group P2₁2₁2₁P6₁ P2₁2₁2₁ P2₁2₁2₁ Cell dimensions a, b, c (Å) 85.0420, 200.0670,86.7165, 86.9260, 122.6570, 200.0670, 123.3500, 123.4100, 212.406096.4100 211.4530 211.4160 Resolution (Å) 40-2.70 (2.78-2.71)* 40-3.25(3.32-3.25) 40-3.5 (3.69-3.5) 40-3.5 (3.69-3.5) R_(sym) or R_(merge)10.7 (48.1) 14.9 (42.6) 14.5 (41.7) 16.1 (43.7) I/σI 9.3 (1.7) 6.4 (2.4)7.0 (3.5) 7.3 (3.6) Completeness (%) 96.97 (95.84) 98.85 (98.1) 85.7(83.1) 93.8 (94.2) Redundancy 4.2 (4.2) 2.8 (2.5) 4.7 (4.8) 5.3 (5.3)Refinement Resolution (Å) 20-2.71 20-3.25 No. reflections 56173 32773R_(work/)R_(free) 23.8/27.7 24.3/29.6 No. atoms Protein 4982 4982 Water358 77 B-factors Protein 52.5 37.8 Water 41.3 26.5 R.m.s deviations Bondlengths (Å) 0.007 0.006 Bond angles (°) 0.848 0.735 Ramachandran plot(%) Most favored 83.3 86.4 Allowed 15.2 11.5 Generously allowed 1.4 1.7Disallowed 0.2 0.4 *Highest resolution shell is shown in parenthesis.

Structure Determination and Refinement.

Initial phases for the orthorhombic crystals were obtained using themethod of single isomorphous replacement with anomalous signal (SIRAS)using two datasets collected from two different mercury (CH₃HgCl)derivatized crystals at two different wavelengths (Hg1—1.00850 Å;Hg2—1.00800 Å) (Table 5). The derivatives were prepared by soaking thecrystals with 5 mM methyl mercury chloride (CH₃HgCl) for 15 minutes.Initially, twelve heavy atom sites were located using SOLVE (Terwilliger(2003) Methods Enzymol. 374:22-37) and refined and new phases calculatedwith MLPHARE (Collaborative Computational Project 4 (1994) ActaCrystallogr. D 50:760-763). MLPHARE improved phases were used toidentify the remaining heavy atom sites (twenty two in total) bycalculating an anomalous difference map to 3.5 Å resolution. MLPHAREphases obtained using all the heavy atom sites where then used in DMwith two-fold NCS and phase extension using the high-resolution (2.71 Å)dataset collected, at 1.00800 Å wavelength, to calculate startingexperimental maps. These maps were sufficiently good for model buildingwhich was carried out in COOT (Emsley & Cowtan (2004) Acta Crystallogr DBiol Crystallogr 60:2126-32). The electron density map revealed cleardensity for all 596 residues of the protein. However, density for thenucleic acid substrate in the structure was not observed. The model wasrefined using both CNS-SOLVE (Brunger, et al. (1998) Acta Crystallogr DBiol Crystallogr 54:905-21) and REFMAC5 (Murshudov, et al. (1997) ActaCrystallogr D Biol Crystallogr 53:240-55). The last cycles of refinementwere carried out with TLS restraints as implemented in REFMAC5 (Table5). The P2₁2₁2₁ refined model was used to solve the structure of theTERT crystallized in the P6₁ crystal form (data collected at 0.97980 Åwavelength) by molecular replacement with PHASER (Potterton, et al.(2003) Acta Crystallogr D Biol Crystallogr 59:1131-7).

Architecture of the TERT Structure.

The structure of the full-length catalytic subunit of the T. castaneumactive telomerase, TERT, was determined to 2.71 Å resolution. Asindicated, there was a dimer in the asymmetric unit (AU), however theprotein alone was clearly monomeric in solution as indicated by gelfiltration and dynamic light scattering, indicating that the dimerobserved in the crystal was the result of crystal packing. This wasfurther supported by the fact that a different crystal form (Table 5) ofthe same protein also contained a dimer in the AU of differentconfiguration. It is worth noting that the TERT from this organism doesnot contain a TEN domain, a low conservation region of telomerase (FIG.1B).

The TERT structure is composed of three distinct domains, a TER-bindingdomain (TRBD), the reverse transcriptase (RT) domain, and the C-terminalextension thought to represent the putative “thumb” domain of TERT(FIGS. 1A and 1B). As indicated herein, the TRBD is mostly helical andcontains an indentation on its surface formed by two conserved motifs(CP and T) which bind double- and single-stranded RNA, respectively, andhas been defined as the template boundary element of the RNA substrateof telomerase, TER. Structural comparison of the TRBD domain from T.castaneum with that of the structure from T. thermophila shows that thetwo structures are similar (RMSD 2.7 Å), indicating a high degree ofstructural conservation between these domains across organisms ofdiverse phylogenetic groups.

The RT domain is a mix of α-helices and β-strands organized into twosubdomains that are most similar to the “fingers” and “palm” subdomainsof retroviral reverse transcriptases such as HIV reverse transcriptase(PDB code ID 1N5Y; Sarafianos, et al. (2002) EMBO J. 21:6614-24), viralRNA polymerases such as hepatitis C viral polymerase (Code ID 2BRL DiMarco, et al. (2005) J. Biol. Chem. 280:29765-70) and B-family DNApolymerases such as RB69 (PDB Code ID 1WAF; Wang, et al. (1997) Cell89:1087-99), and contain key signature motifs that are hallmarks ofthese families of proteins (Lingner, et al. (1997) Science 276:561-7)(FIGS. 3A-3C). Structural comparison of TERT with the HIV RTs, showsthat the “fingers” subdomain of TERT (i.e., motifs 1 and 2) are arrangedin the open configuration with respect to the “palm” subdomain (i.e.,motifs A, B′, C, D, and E), which is in good agreement with theconformation adopted by HIV RTs in the absence of bound nucleotide andnucleic acid substrates (Ding, et al. (1998) J. Mol. Biol.284:1095-111). One striking difference between the putative “palm”domain of TERT and that HIV reverse transcriptases is a long insertionbetween motifs A and B′ of TERT referred to as the IFD motif that isrequired for telomerase processivity (Lue, et al. (2003) Mol. Cell.Biol. 23:8440-9). In the TERT structure, the IFD insertion is composedof two anti-parallel α-helices (α13 and α14) located on the outsideperiphery of the ring and at the interface of the “fingers” and the“palm” subdomains. These two helices are almost in parallel positionwith the central axis of the plane of the ring and make extensivecontacts with helices α10 and α15 and play an important role in thestructural organization of this part of the RT domain. A similarstructural arrangement is also present in viral polymerases, and theequivalent of helix α10 in these structures is involved in directcontacts with the nucleic acid substrate (Ferrer-Orta, et al. (2004) J.Biol. Chem. 279:47212-21).

In contrast to the RT domain, the C-terminal extension is an elongatedhelical bundle that contains several surface exposed, long loops. Asearch in the protein structure database using the software SSM(Krissinel & Henrick (2004) Acta Cryst. D60:2256-2268; Krissinel (2007)Bioinformatics 23:717-723) produced no structural homologues suggestingthat the CTE domain of telomerase adopts a novel fold. Structuralcomparison of TERT with the HIV RT, the viral RNA polymerases andB-family DNA polymerases places the “thumb” domain of these enzymes andthe CTE domain of TERT in the same spatial position with respect to the“fingers” and “palm” subdomains, indicating that the CTE domain oftelomerase is the “thumb” domain of the enzyme, a finding that is ingood agreement with previous biochemical studies (Hossain, et al. (2002)J. Biol. Chem. 277:36174-80).

TERT domain organization brings the TRBD and “thumb” domains, whichconstitute the terminal domains of the molecule, together, anarrangement that leads to the formation of a ring-like structure that isreminiscent of the shape of a donut (FIG. 1C). Several lines of evidenceindicate that the domain organization of the TERT structure presentedherein is biologically relevant. First, the domains of four TERTmonomers observed in two different crystal forms (two in each asymmetricunit) are organized the same (average RMSD=0.76 Å between all fourmonomers). Second, contacts between the N- and C-terminal domains ofTERT are extensive (1677 Å²) and largely hydrophobic in nature involvingamino acid residues Tyr4, Lys76, Thr79, Glu84, Ser81, His87, Asn142,His144, Glu145, Tyr411, His415, Phe417, Trp420, Phe422, Ile426, Phe434,Thr487, Ser488, Phe489, and Arg592. This observation is in agreementwith previous biochemical studies (Arai, et al. (2002) J. Biol. Chem.277:8538-44). Third, TERT domain organization is similar to that of thepolymerase domain (p66 minus the RNase H domain) of its closesthomologue, HIV reverse transcriptase (Sarafianos, et al. (2002) supra),the viral RNA polymerases (Di Marco, et al. (2005) supra) and theB-family DNA polymerases and in particular RB69 (Wang, et al. (1997)supra). The arrangement of the TERT domains creates a hole in theinterior of the particle that is ˜26 Å wide and ˜21 Å deep, sufficientto accommodate double-stranded nucleic acids approximately seven toeight bases long, which is in good agreement with existing biochemicaldata (Forstemann & Lingner (2005) EMBO Rep. 6:361-6; Hammond & Cech(1998) Biochemistry 37:5162-72).

The TERT Ring Binds Double-Stranded Nucleic Acid.

To understand how the TERT ring associates with RNA/DNA to form afunctional elongation complex, a double-stranded nucleic acid wasmodeled into the interior using the HIV reverse transcriptase—DNAcomplex (Sarafianos, et al. (2002) supra), TERT's closest structuralhomologue. The TERT-RNA/DNA model immediately showed some strikingfeatures that supported the model of TERT-nucleic acid associations. Thehole of the TERT ring and where the nucleic acid heteroduplex wasprojected to bind was lined with several key signature motifs that arehallmarks of this family of polymerases and have been implicated innucleic acid association, nucleotide binding and DNA synthesis.Moreover, the organization of these motifs resulted in the formation ofa spiral in the interior of the ring that resembled the geometry of thebackbone of double-stranded nucleic acid. Several of the motifs,identified as contact points with the DNA substrate, were formed partlyby positively charged residues, the side chains of which extended towardthe center of the ring and were poised for direct contact with thebackbone of the DNA substrate. For example, the side chain, of thehighly conserved K210 that forms part of helix α10, is withincoordinating distance of the backbone of the modeled DNA thus providingthe stability required for a functional telomerase enzyme. Helix α10lies in the upper segment of the RT domain and faces the interior of thering. The location and stabilization of this helix is heavily influencedby its extensive contacts with the IFD motif implicated in telomeraseprocessivity (Lue, et al. (2003) Mol. Cell. Biol. 23:8440-9). Disruptionof the IFD contacts with helix α10 through deletion or mutations of thismotif would lead to displacement of helix α10 from its current location,which would in turn effect DNA-binding and telomerase function.

Structural elements of the “thumb” domain that localized to the interiorof the ring also made several contacts with the modeled DNA substrate.In particular, the loop (“thumb” loop) that connects the “palm” to the“thumb” domain and constitutes an extension of the E motif also known asthe “primer grip” region of telomerase, preserves to a remarkabledegree, the geometry of the backbone of double stranded nucleic acid.The side chains of several lysines (e.g., Lys406, Lys416, Lys418) andasparagines (e.g., Asn423) that formed part of this loop extended towardthe center of the TERT molecule and were within coordinating distance ofthe backbone of modeled double-stranded nucleic acid. Of particularinterest was Lys406. This lysine was located in proximity of motif E andits side chain extended toward the nucleic acid heteroduplex and waspoised for direct contacts with the backbone of the nucleotides locatedat the 3′ end of the incoming DNA primer. It is therefore possible thatthe side chain of this lysine together with motif E help facilitateplacement of the 3′-end of the incoming DNA substrate at the active siteof the enzyme during telomere elongation. Sequence alignments of the“thumb” domain of TERTs from a wide spectrum of phylogenetic groupsshowed that the residues predicted to contact the DNA substrate arealways polar (FIGS. 3A-3C). Another feature of the “thumb” domain thatsupported double-stranded nucleic acid binding was helix α19, a 3¹⁰helix (“thumb” 3¹⁰ helix) that extended into the interior of the ringand appeared to dock itself into the minor groove of the modeleddouble-stranded nucleic acid thus facilitating RNA/DNA hybrid bindingand stabilization. Deletion or mutation of the corresponding residues inboth yeast and human TERT results in sever loss of TERT processivityclearly indicating the important role of this motif in TERT function(Hossain, et al. (2002) J. Biol. Chem. 277:36174-80; Huard, et al.(2003) Nucleic Acids Res. 31:4059-70; Banik, et al. Mol. Cell. Biol.22:6234-46).

The Active Site of TERT and Nucleotide Binding.

The T. castaneum TERT structure presented herein was crystallized in theabsence of nucleotide substrates and magnesium, however, the locationand organization of TERT's active site and nucleotide binding pocket wasdetermined on the basis of existing biochemical data (Lingner, et al.(1997) supra) and structural comparison with the polymerase domain ofits closest homologue, the HIV reverse transcriptase (Das, et al. (2007)J. Mol. Biol. 365:77-89). The TERT active site is composed of threeinvariant aspartic acids (Asp251, Asp343 and Asp344) that form part ofmotifs A and C, two short loops located on the “palm” subdomain, andadjacent to the “fingers” of TERT. Structural comparison of TERT withHIV reverse transcriptases, as well as RNA and DNA polymerases showed ahigh degree of similarity between the active sites of these families ofproteins indicating that telomerase also employs a two-metal mechanismfor catalysis. Alanine mutants of these TERT aspartic acids resulted incomplete loss of TERT activity indicating the essential role of theseresidues in telomerase function (Lingner, et al. (1997) supra).

The telomerase nucleotide binding pocket is located at the interface ofthe “fingers” and “palm” subdomains of TERT and is composed of conservedresidues that form motifs 1, 2, A, C, B′ and D implicated in templateand nucleotide binding (Bosoy & Lue (2001) J. Biol. Chem. 276:46305-12;Haering, et al. (2000) Proc. Natl. Acad. Sci. USA 97:6367-72).Structural comparisons of TERT with viral HIV reverse transcriptasesbound to ATP (Das, et al. (2007) supra) supports nucleotide substrate inthis location. Two highly conserved, surface-exposed residues Tyr256 andVal342 of motifs A and C, respectively, form a hydrophobic pocketadjacent to and above the three catalytic aspartates and couldaccommodate the base of the nucleotide substrate. Binding of thenucleotide in this oily pocket places the triphospate moiety inproximity of the active site of the enzyme for coordination with one ofthe Mg²⁺ ions while it positions the ribose group within coordinatingdistance of an invariant glutamine (Gln308) that forms part of motif B′thought to be an important determinant of substrate specificity (Smith,et al. (2006) J. Virol. 80:7169-78). Protein contacts with thetriphospate moiety of the nucleotide are mediated by motif D, a longloop, located beneath the active site of the enzyme. In particular, theside chain of the invariant Lys372 is within coordinating distance ofthe γ-phosphate of the nucleotide an interaction that most likely helpsposition and stabilize the triphosphate group during catalysis. The sidechains of the highly conserved Lys189 and Arg194 of motifs 1 and 2,which together form a long β-hairpin that forms part of the “fingers”subdomain, are also within coordinating distance of the both the sugarand triphosphate moieties of the modeled nucleotide. Contacts witheither or both the sugar moiety and the triphosphate of the nucleotidesubstrate would facilitate nucleotide binding and positioning forcoordination to the 3′-end of the incoming DNA primer.

TRBD Facilitates Template Positioning at the Active Site of TERT.

As with most DNA and RNA polymerases, nucleic acid synthesis bytelomerase requires pairing of the templating region (usually seven toeight bases or more) of TER with the incoming DNA primer (Lee &Blackburn (1993) Mol. Cell. Biol. 13:6586-99). TRBD-RT domainorganization forms a deep cavity on the surface of the protein thatspans the entire width of the wall of the molecule, forming a gap thatallows entry into the hole of the ring from its side. The arrangement ofthis cavity with respect to the central hole of the ring provides anelegant mechanism for placement of the RNA template, upon TERT-TERassembly, in the interior of the ring and where the enzyme's active siteis located. Of particular significance is the arrangement of theβ-hairpin that forms part of the T-motif. This hairpin extends from theRNA-binding pocket and makes extensive contacts with the “thumb” loopand motifs 1 and 2. Contacts between this hairpin and both the “fingers”and the “thumb” domains place the opening of the TRBD pocket that facesthe interior of the ring in proximity to the active site of the enzyme.It is therefore likely that this β-hairpin acts as an allostericeffector switch that couples RNA-binding in the interior of the ring andplacement of the RNA template at the active site of the enzyme.Placement of the template into the interior of the molecule wouldfacilitate its pairing with the incoming DNA substrate, which togetherwould form the RNA/DNA hybrid required for telomere elongation. RNA/DNApairing is a prerequisite of telomere synthesis in that it brings the3′-end of the incoming DNA primer in proximity to the active site of theenzyme for nucleotide addition while the RNA component of theheteroduplex provides the template for the faithful addition ofidentical repeats of DNA at the ends of chromosomes. Strikingly,modeling of the RNA/DNA heteroduplex in the interior of the TERT ringplaces the 5′-end of the RNA substrate at the entry of the RNA-bindingpocket and where TERT is expected to associate with TER while it placesthe 3′-end of the incoming DNA primer at the active site of TERTproviding a snapshot of the organization of a functional telomeraseelongation complex.

Example 3 Structure of T. castaneum Telomerase Catalytic Subunit TERTBinding to RNA Template and Telomeric DNA

Methods for Expressing and Purifying Proteins.

Wild-type and mutant (Asp251Ala) T. castaneum, full length TERT proteinswere overexpressed and purified as described herein with subtlemodifications that increased protein yield. Specifically, the proteinswere over-expressed in E. coli Rosetta (DE3) pLysS (Novagen) at 30° C.for 5 hours with slow shaking (shaker/incubator setting, 120 rpm). Stockprotein (10 mg/ml) was dialyzed in 10 mM Tris-HCl, 100 mM KCl, 1 mMTCEP, pH 7.5 prior to crystallization trials.

Methods for Preparing T. castaneum Extracts and Isolating RNA.

Twenty T. castaneum larvae or pupae were ground in liquid N₂,homogenized with 200 μl of extraction buffer (25 mM Tris-HCl, 5 mMβ-mercaptoethanol ((3-ME), 1 mM EGTA, 0.1 mM benzamidine, 200 mM KCl,10% (w/v) glycerol, 10 mM imidazole and RNasin (PROMEGA, Madison, Wis.),pH 7.5) and placed on ice for 30 minutes. After the homogenate wascentrifuged at 12,000 g at 4° C. for 20 minutes, the supernatant wascollected, flush frozen in liquid nitrogen and stored at −80° C. beforeuse. The total RNA was then extracted from the T. castaneum homogenateusing the RNEASY Protect Mini Kit (QIAGEN, Valencia, Calif.).

Methods for In Vitro Reconstitution of T. castaneum Telomerase.

The telomerase RNA of T. castaneum had not been previously identified.Therefore, total RNA was isolated from beetle larvae and used with therecombinant TERT to assemble the telomerase complex in vitro. Twenty μgof the his-tagged TERT (25 mM Tris, 200 mM KCl, 10% (w/v) glycerol, 5 mMβ-ME and 10 mM imidazole, pH 7.5) was mixed with 50 μl of T. castaneumlarvae total RNA and the two were incubated in T. castaneum lysate fortwo hours at room temperature in the presence of RNasin. The telomerasecomplex was then purified over a Ni-NTA column and tested for activityusing a modified TRAP assay (Sasaki & Fujiwara (2000) Eur. J. Biochem.267:3025-3031) as described herein.

Telomerase Repeat Amplification Protocol (TRAP) Assays.

The activity of the in vitro reconstituted T. castaneum telomerase wastested using the following TRAP assay. The telomerase elongation stepwas carried out in a 50 μl reaction mixture composed of 20 mM Tris-HCl(pH 8.3), 7.5 mM MgCl₂, 63 mM KCl, 0.05% (w/v) TWEEN 20, 1 mM EGTA,0.01% (w/v) BSA, 0.5 mM of each dNTP, 1 μM DNA primer (5′-aag ccg tcgagc aga gtc-3′; SEQ ID NO:17) (Tcas-TS)) and 4 μg of Ni-NTA purified, T.castaneum telomerase. After incubation at 30° C. for 60 minutes, eachreaction mixture was phenol-chloroform extracted and precipitated withethanol. Each sample was re-suspended in 50 μl of PCR reaction buffer(10 mM Tris/HCl (pH 8.0), 50 mM KCl, 2 mM MgCl₂, 100 μM dNTPs (dATP dTTPdGTP), 10 μM [³²P]dCTP (80 Ci per mmol), 1 μM Tcas-CX primer (5′-gtg tgacct gac ctg acc-3′; SEQ ID NO:18) and HOTSTARTAQ DNA polymerase(QIAGEN). The PCR products were resolved on Tris-borate-EDTA(TBE)-polyacrylamide gels. The presence of multiple telomeric repeats(TCAGG)_(n) was also confirmed by subcloning and sequencing the TRAPproducts.

RNA-DNA Hairpins.

The RNA-DNA hairpins tested for TERT binding, activity and subsequentlyfor structural studies are shown in Table 6. All three hairpins containthe putative RNA-templating region (5′-rCrUrGrArCrCrU-3′) (one and ahalf times the telomeric repeat of T. castaneum, TCAGG) and thecomplementary telomeric DNA sequence (5′-GTCAGGT-3′). The RNA-templatingregion and the DNA complement were connected for stability with anRNA-DNA linker (loop). The hairpins were designed to contain a 5′-RNAoverhang so that the telomerase complex could be trapped in itsreplication state upon co-crystallization with the complementarynon-hydrolysable nucleotide(s). The only difference between the threehairpins is the length of the linker.

TABLE 6 Hairpin Sequence (5′->3′) SEQ ID NO: 21 mer_(r)C_(r)U_(r)G_(r)A_(r)C_(r)C_(r)U _(r)G_(r)A_(r)CTTCGGTCAGGT 19 17 mer_(r)C_(r)U_(r)G_(r)A_(r)C_(r)C_(r)U _(r)GTTCGCAGGT 20 15 mer_(r)C_(r)U_(r)G_(r)A_(r)C_(r)C_(r)U TTCG AGGT 21 *Underlined sequencesrepresent linker sequences. Bold sequences form the loop of the hairpin.

TERT, Reverse Transcriptase (RT) Assays.

Standard reverse transcriptase assays were carried out using therecombinant T. castaneum TERT and the RNA-DNA hairpin (Table 6) to testTERT's ability to replicate the end of the DNA substrate that includespart of the RNA-DNA hairpin. RT assays were carried out in telomerasebuffer (50 mM Tris-HCl, 100 mM KCl, 1.25 mM MgCl₂, 5 mM DTT, 5% (w/v)glycerol, pH 8 at room temperature), 100 μM dNTPs (dATP dTTP dGTP), 10μM [³²P]dCTP (80 Ci per mmol), 5 μM RNA-DNA hairpin and 1 μM recombinantTERT. The samples were incubated for two hours at room temperature, thenphenol/chloroform extracted and ethanol precipitated. The DNA pellet wasre-suspended in a solution composed of 90% (w/v) formamide and 10% (w/v)glycerol and the sample was run on a 12% (w/v) polyacrylamide-7 M ureagel in 1×TBE at 220V for 70 minutes at 4° C.

Protein Crystallization and Data Collection.

The binary complex was prepared by adding to the dialyzed protein 1.2molar excess nucleic acid (RNA-DNA hairpin purchased from Integrated DNATechnologies), 5 mM dNTPαS (Jena Biosciences GmbH) and 5 mM MgCl₂.Crystals of the monoclinic space group P2₁ that diffracted to 2.7 Åresolution appeared in three days and grew to final size in two weeks.Crystals were grown by the vapor diffusion, sitting drop method bymixing one volume of the ternary complex with one volume of reservoirsolution containing 0.1 M HEPES, (pH 7.5) and 12% 1,6-hexanediol or PEG4K and 0.2 M KCl. Crystals were transferred into cryoprotectant solutionthat contained 0.1 M HEPES (pH 7.5), 15% (w/v) 1,6-hexanediol or PEG 4K,15% (w/v) glycerol, 0.2 M KCl and 1 mM TCEP and were harvested by flashfreezing in liquid nitrogen. Data were collected at the NSLS, beam lineX25 and processed with MOSFILM as implemented in WEDGER-ELVES (Holton &Alber (2004) Proc. Natl. Acad. Sci. USA 101:1537-1542) (Table 7). Thereis one monomer in the asymmetric unit.

TABLE 7 TERT Complex Data Collection Space group P2₁ Cell dimensions a,b, c (Å) 77.2 52.8 101.6 α, β, γ (°) 90 101.9 90 Resolution (Å) 20-2.7(2.85-2.70)* R_(sym) or R_(merge) 11.4 (45.8) I/σI 7.9 (2.3)Completeness (%) 93.7 (96.1) Redundancy 2.8 (2.5) Refinement Resolution(Å) 20-2.7 No. Reflections 19815 R_(work)/R_(free) 24.2/28.7 Number ofatoms 4982 Protein 4982 Ligand/Ion 496/1  Water 54 B-factors Protein 45Ligand/Ion 37 Water 21 R.m.s. Deviations Bond lengths (Å) 0.006 Bondangles (°) 0.887 *Number of crystals used - one. *Values in parenthesesare for highest-resolution shell.

Structure Determination and Refinement.

Phases where calculated by molecular replacement (MR) using PHASER(Potterton, et al. (2003) Acta Crystallogr D Biol Crystallogr59:1131-1137) as implemented in CCP4 suit of programs using thesubstrate-free TERT structure described herein as a search model. Mapscalculated after one cycle of refinement by CNS-SOLVE revealed clear2fo-fc density for all 596 residues of TERT and fo-fc density for thenucleic acid substrate at 3.0 σ contour level. Model building wascarried out in COOT (Emsley & Cowtan (2004) Acta Crystallogr D BiolCrystallogr 60:2126-2132) and the model was refined using both CNS-SOLVE(Brunger, et al. (1998) Acta Crystallogr. D Biol. Crystallogr.54:905-921) and REFMAC5 (Murshudov, et al. (1997) Acta Crystallogr. DBiol. Crystallogr. 53:240-255). The last cycles of refinement werecarried out with TLS restraints as implemented in REFMAC5. The structurewas refined to good stereochemistry with 84.3, 14.3 and 1.4 of theresidues in the most favorable, additional allowed and generouslyallowed of the Ramachandran plot, respectively.

TERT Structure Overview. The full length (FIG. 1B), active, T.castaneum, TERT was co-crystallized with an RNA-DNA hairpin containingthe putative RNA templating region (5′-rCrUrGrArCrCrU-3′) and thecomplementary telomeric DNA (5′-GTCAGGT-3′) joined together with a shortRNA-DNA linker (Table 6). It is worth noting that the T. castaneum TERTlacks the TEN domain, required for activity and processivity in severaleukaryotic telomerase genes, including human (Wyatt, et al. (2009) PLoSOne 4:e7176) and Tetrahymena thermophila (Zaug, et al. (2008) Nat.Struct. Mol. Biol. 15:870-872; Finger & Bryan (2008) Nucleic Acids Res.36:1260-1272). The absence of the TEN domain from T. castaneum couldexplain the reduced activity observed for this enzyme when compared tothose containing this domain. The RNA component of T. castaneumtelomerase has not been previously identified. Therefore, the followinginformation was used to predict its templating region: The RNAtemplating region of telomerase is usually one and a half telomericrepeats (Lee & Blackburn (1993) Mol. Cell. Biol. 13: 6586-6599; Lingner,et al. (1994) Genes Dev. 8: 1984-1998; Shippen-Lentz & Blackburn (1990)Science 247: 546-552) and this sequence is known for many organisms(telomerase database). For example the mammalian telomerase templatingregion is “CUAACCCU” and the telomeric repeat is “TTAGGG”. The telomericrepeat for T. castaneum is “TCAGG” (Osanai, et al. (2006) Gene376:281-89; Richards, et al. (2008) Nature 452:949-955). The RNA-DNAhairpin was designed to contain a three-nucleotide overhang at the5′-end of the RNA template, so that the enzyme could be trapped in itscatalytic state upon co-crystallization of the protein-nucleic acidassembly with Mg⁺⁺ ions and non-hydrolysable nucleotides. In an effortto identify a hairpin suitable for crystallographic studies, a number ofRNA-DNA hairpins were screened (15mer, 18mer and 21mer; Table 6) wherethe templating region and the complementary DNA sequence were kept thesame but the length of the linker was changed. Although, all threehairpins tested had the same binding affinity for TERT and reversetranscriptase activity, only the 21mer was amenable to crystallographicstudies. It was noted that in the structure, the hairpin linker extendsout of the TERT ring and is only involved in crystal contacts withadjacent molecules. The crystals were also grown in the presence of theslowly hydrolysable nucleotide analogues dNTPαS and Mg⁺⁺ ions.

The structure was solved at 2.7 Å resolution using the method ofmolecular replacement with the substrate-free TERT described herein as asearch model. All of the TERT molecule and the RNA-DNA hybrid wereinterpretable in electron density maps. Unexpectedly, there was extradensity for three nucleotides at the 3′-end of the telomeric DNA,indicating that TERT had extended the 3′-end of the DNA substrate in thecrystallization drop. There was no evidence for nucleotide at the activesite of the enzyme, which was partially occupied by the nucleotidelocated at the 3′-end of the DNA substrate. Several lines of evidenceindicate that the association of the RNA templating region and the DNAsubstrate with TERT in the structure presented here are biologicallyrelevant. First, TERT is an active polymerase in the presence of thenucleic acid substrate used in this study both by standard reversetranscriptase assays and in the crystallization drop. Second, the RNAtemplate makes contacts with conserved motifs that are hallmarks of thisfamily of enzymes. Third, contacts between TERT and the RNA templateposition the solvent accessible bases adjacent to and above the activesite of the enzyme for nucleotide binding thus facilitating selectivity.Moreover, TERT-RNA associations position the 5′-end of the RNA-templateat the entry of the TRBD RNA-binding pocket and where the templateboundary element located upstream of the RNA-template in many organismsis thought to bind. Fourth, interactions between the DNA substrate andthe primer grip region (a characteristic shared among telomerase and HIVRTs) place the 3′-end of the DNA substrate at the active site of theenzyme where it is accessible for nucleotide addition. Fifth,TERT-nucleic acid associations are strikingly similar to those observedof HIV reverse transcriptase, TERT's closest structural homologue.

The four major TERT domains: the RNA-binding domain (TRBD); the fingersdomain, implicated in nucleotide binding and processivity (Bosoy & Lue(2001) J. Biol. Chem. 276:46305-46312); the palm domain, which containsthe active site of the enzyme; and the thumb domain, implicated in DNAbinding and processivity (Hossain, et al. (2002) J. Biol. Chem.277:36174-80), were organized into a ring configuration similar to thatobserved for the substrate-free enzyme described herein (FIG. 1C). Thearrangement of the TERT domains created a highly positively chargedcavity in the interior of the TERT ring, which was 22 Å wide and 21 Ådeep and could accommodate seven bases of double stranded nucleic acid.Within this cavity binds one molecule of the RNA-DNA hybrid, whichassembles together, via Watson-Crick base pairing into a helicalstructure similar to both the DNA-DNA and RNA-DNA structure bound to HIVreverse transcriptase (Huang, et al. (1998) Science 282:1669-75;Sarafianos, et al. (2001) EMBO J. 20:1449-61).

TERT Nucleic Acid Associations.

Structure analysis indicated that interactions between the protein andthe RNA templating region were mediated by the fingers, the palm andthumb domains. The 5′-end RNA cytosine (rC1) and uracil (rU2) werelocated at the interface of the fingers and palm domains and wereinvolved in a network of interactions with conserved residues (FIG. 3)of motifs 2 and B′ both of which are located in proximity of the activesite of the enzyme. In particular, the 2′-OH and the base carbonyl ofrC1 is within hydrogen bonding distance of the backbone carbonyls ofVal197 of motif 2 and Gly309 of motif B′ while the pyrimidine base sitsover the otherwise solvent exposed, hydrophobic side chain of theconserved Ile196 that also forms part of motif 2 (FIG. 5). Contactsbetween rU2 and the protein are mediated by the short aliphatic sidechain of Pro311 and the ribose group (FIG. 5). Interactions between rC1and rU2 with motifs 2 and B′ place the cytosine base in proximity of theactive site of the enzyme, where it is well-positioned for Watson-Crickbase-pairing with the incoming nucleotide substrate. Stabilization andplacement of the 5′-end bases of the templating region above the activesite of the enzyme was in large part mediated by the interactions of theremaining five ribonucleotides with the incoming DNA primer. Limitedcontacts between this part of the RNA and the protein were mediated viaa water molecule (Wat18) which coordinates the 2′-OH of guanosine (rG3)with the backbone of helix α15 (FIG. 5). Notably, the structuralorganization of helix α15 was influenced by the IFD motif, a longinsertion, composed of two helices (α13 and α14), between motifs A andB′, which explains why mutations in this motif lead to loss oftelomerase function (Lue, et al. (2003) Mol. Cell. Biol. 23:8440-49).

Contacts between TERT and the DNA substrate were mediated in large partvia backbone interactions with the thumb loop and helix. The thumbhelix, as described herein, sits in the minor groove of the RNA-DNAheteroduplex, making extensive contacts with the phosphodiester backboneand the ribose groups of the RNA-DNA hybrid. The mode of action of thethumb helix of telomerase appears to be similar to that proposed for theequivalent helix (helix H) of retroviral reverse transcriptases(Jacobo-Molina, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6320-24;Kohlstaedt, et al. (1992) Science 256:1783-1790). Another conservedelement of the thumb domain, known as the thumb loop, runs almostparallel to the curvature of the DNA primer and the two are involved ina network of backbone and solvent mediated interactions (FIG. 6).Interactions between the DNA and the thumb loop included the side chainsof Lys416 and Asn423, both of which extended toward the center of thering and were within hydrogen bonding distance of the DNA backbone.Contacts between the thumb domain and the DNA position the nucleotideslocated at its 3′-end within coordinating distance of the primer gripregion (motif E), a short, rigid loop located at the interface of thepalm and thumb domains, and in proximity of the active site of theenzyme (FIG. 7). The backbone of the tip of this loop formed by theconserved residues Cys390 and Gly391 abuts the ribose group of C22 andthis interaction guides the 3′-end DNA nucleotides toward the activesite of the enzyme (FIG. 7). The active site of the enzyme, and wherethe incoming nucleotide is projected to bind, is partially occupied bythe nucleotide (G24) located at the 3′-end of the DNA (FIG. 7). Theribose group, and to a certain extend the guanosine base of G24, whichmakes Watson-Crick pairing interactions with the rC1 located at the5′-end of the RNA template, sit in a well-defined hydrophobic pocketformed by the side chains of the invariant Tyr256, Gln308 and conservedVal342 of motifs A, B′ and C respectively, while the α-phosphate iscoordinated by the magnesium ion occupying the active site aspartates(FIG. 7). The important role of Val342 in telomerase selectivity hasbeen shown for the human telomerase holoenzyme (Drosopoulos & Prasad(2007) Nucleic Acids Res. 35:1155-1168).

TERT Domain Rearrangements Upon Nucleic Acid Binding.

Comparisons of the nucleic acid bound and substrate-free TERT structuresindicate that TERT nucleic acid associations induce small rigid-bodychanges in orientation between subunits of the enzyme that lead to a 3.5Å decrease of the diameter of the interior cavity of the ring. Thedecrease in the diameter of the central cavity arises from a 6° inwardrotation together with a 3.5 Å translation, of the thumb domain withrespect to the fingers and palm domains. Translation of the thumb domaintoward the center of the ring is accompanied by the TRBD, which is alsoshifted 3.5 Å toward the finger domain creating a more narrowRNA-binding pocket than the substrate-free enzyme. The role of thissubtle structural rearrangement may have implications for TERTassociation with the full-length RNA, TER.

Common Aspects of Substrate Binding Between TERT and HIV RTs.

It has been postulated that telomerase uses a mechanism of DNAreplication that resembles that of other retroviral reversetranscriptases, a suggestion supported by the structure of theTERT-nucleic acid complex presented here. Structural comparison of theRNA-DNA bound TERT and HIV RT (PDB ID: 1HYS36) shows a strikingsimilarity in the overall domain organization and nucleic acid bindingbetween the two structures. Like HIV RTs, telomerase-dependent telomerereplication requires pairing of the templating region with the incomingDNA primer and placement of the 3′-end of the DNA into the enzyme'sactive site for nucleotide addition. Moreover, TERT- or HIV RT-nucleicacid associations are accompanied by domain rearrangements thatfacilitate the formation of a tight, catalytic, protein nucleic acidassembly and positioning of the DNA 3′-end at the active site of theenzyme for catalysis (Kohlstaedt, et al. (1992) supra; Rodgers, et al.(1995) Proc. Natl. Acad. Sci. USA 92:1222-1226; Steitz (1997) HarveyLect. 93:75-93). Contacts between the protein and the RNA templatingregion are specific and involve telomerase key signature motifs (motif 2and B′ of the fingers and palm domains, respectively) that are hallmarksof these families of enzymes and are required for positioning of thesolvent accessible bases of the RNA template in proximity of the activesite for nucleotide binding and selectivity. Contacts between theprotein and the DNA substrate are mediated by the thumb domain anddespite the lack of sequence homology in this region between the twofamilies of enzymes, the mode of action of the thumb helix of telomeraseis similar to that proposed for helix H of HIV RTs (Jacobo-Molina, etal. (1993) supra; Kohlstaedt, et al. (1992) supra; Beese, et al. (1993)Science 260: 352-355). Placement of the DNA 3′-end at the active site ofthe enzyme is further facilitated by the primer grip region anotherhighly conserved motif between TERT and HIV RTs44 (FIG. 7).

Although the enzyme was not trapped in its catalytic state, thepartially occupied active site of the enzyme, formed by a number ofinvariant (Asp251, Tyr256, Gln308, Asp343, Asp344) or highly conservedresidues (Val342), by the nucleotide G24 located at the 3′-end of theDNA (FIG. 7) suggests the mechanism of nucleotide binding andselectivity of telomerase during the replication process. In a similarmanner, the invariant Tyr256 and Gln308 are also present in HIV RTswhere, like in TERT, they are involved in nucleotide binding andselectivity through positioning for interactions with the templatingregion (Huang, et al. (1998) supra; Cases-Gonzalez, et al. (2000) J.Biol. Chem. 275:19759-19767), further supporting common mechanisticaspects of DNA replication between these families of enzymes.

TERT Rigid Conformational Changes and Function.

Domain re-organization, upon nucleic acid binding, are a common featureof RNA-, DNA-polymerases and retroviral reverse transcriptases, and aregeared toward the formation of a tight, catalytic protein nucleic acidassembly and positioning of the DNA 3′-end at the active site of theenzyme for catalysis (Kohlstaedt, et al. (1992) supra; Rodgers, et al.(1995) supra; Steitz (1997) supra). Unlike HIV RTs, telomerase appearsto exist, at least in the absence of the full-length integral RNAcomponent, in a closed ring configuration, an arrangement mediated byextensive contacts between the TRBD and the thumb domains. Comparison ofthe nucleic acid bound and substrate-free TERT structures indicate thatTERT nucleic acid associations induce subtle, rigid-body changes inorientation between subunits of the enzyme that lead to a 3.5 Å decreaseof the diameter of the interior cavity of the ring. These observationswere unexpected because in most polymerases, including the HIV RT, thefingers and thumb domains undergo significant conformational changesrequired for substrate binding and function (Steitz (1997) supra; Ding,et al. (1998) J. Mol. Biol. 284:1095-1111; Steitz (1999) J. Biol. Chem.274:17395-98). For example, the fingers domain, which is known to bindand position the nucleotide at the active site of the enzyme, undergoessignificant conformational changes referred to as the open and closedstates (Ding, et al. (1998) supra). It is therefore possible that theinteractions between the TRBD and the thumb domains lock the fingersdomain in place, thus preventing the conformational rearrangementsobserved in other polymerases, which would indicate the possibility of apreformed active site. A preformed active site has been observed for theHepatitis C viral RNA polymerase (NS5B) (Bressanelli, et al. (2002) J.Virol. 76:3482-92), a close structural homologue of TERT but also theY-family DNA polymerases (Ling, et al. (2001) Cell 107:91-102). Anotherpossibility is that the substrate-free TERT enzyme was trapped in theclosed fingers conformation. Assuming the later is true, significantmovement of the fingers domain of TERT would most likely require thatthe TRBD and the thumb domains are splayed apart. Contacts between theTRBD and the thumb domain are extensive and would require significantenergy to force them apart. This could be accomplished by accessoryproteins that possibly act in a similar manner to that of the slidingclamp loader of DNA polymerases (Jeruzalmi, et al. (2002) Curr. Opin.Struct. Biol. 12:217-224).

Repeat Addition Processivity.

Telomerase, unlike most polymerases, has the ability to add multipleidentical repeats of DNA to the ends of chromosomes, known as repeataddition processivity. This unique characteristic of telomerase has beenattributed in part to the association of the N-terminal portion of TERTwith TER and the telomeric overhang as well as the IFD motif (Lue, etal. (2003) supra). The TEN domain, present in several organisms, and itsweak interaction with the DNA substrate is thought to be a determinantof repeat addition processivity (Wyatt, et al. (2007) Mol. Cell. Biol.27:3226-40; Moriarty, et al. (2004) Mol. Cell. Biol. 24:3720-33), whilemost recently the TRBD and its stable association with TER has also beenshown to be involved in this process (see data presented herein). In thecomplex presented here, the RNA does not directly engage the RNA-bindingpocket of TRBD. The structure shows that TERT-RNA contacts position the5′-end of the templating region at the entry of the RNA-binding pocketof TRBD. This arrangement would place the template boundary element(Chen &7 Greider (2003) Genes Dev. 17:2747-2752; Lai, et al. (2002)Genes Dev. 16:415-20; Tzfati, et al. (2000) Science 288:863-867) presentin most organisms or the short oligonucleotide overhang of rodent TER51within the RNA binding pocket of TRBD. The stable association of TERwith the TRBD would force the enzyme to stall when reaching thenucleotide located at the 5′-end of the RNA template thus preventingreplication beyond this point. Stalling of the enzyme for extendedperiods would lead to destabilization and dissociation of the RNA-DNAheteroduplex and initiation of another round of telomere replication.

Collectively, the data presented here supports common mechanisticaspects of substrate binding and DNA replication between telomerase andHIV reverse transcriptases, indicating an evolutionary link betweenthese families of enzymes. It also provides novel insights into thebasic mechanisms of telomere replication and length homeostasis bytelomerase. Moreover, the structure presented here provides a detailedpicture of the physical contacts between TERT and its nucleic acidsubstrates, information of use in the design of small moleculeinhibitors of telomerase having therapeutic value in the treatment ofcancer and other diseases associated with cellular aging.

Example 4 Modulators of Telomerase Activity

The X-ray crystal structure (complex of TERT with RNA-DNA, nucleotidesand magnesium ions) showed that the organization of the fingers and palmdomains between the substrate-free and substrate-bound TERT molecules ishighly similar, indicating that like the Hepatitis C viral RNApolymerase (NS5B), telomerase has a preformed active site. Thisobservation is surprising since in most polymerases, including the HIVreverse transcriptase, the fingers domain undergoes significantconformational changes referred to as the open and closed states. In HIVRTs, conformational rearrangements of the fingers domain with respect tothe palm domain are essential for nucleotide binding and positioning atthe active site of the enzyme and their absence from telomerase suggestspossible mechanistic differences in nucleotide binding and selectivitybetween these families of enzymes.

The organization of the fingers and palm domains of TERT creates a deep,well-defined narrow mostly hydrophobic Fingers-Palm pocket (FP-pocket;residues Y170, F193, A195, D254, W302, H304, L307, Q308) that opens intothe interior cavity of the TERT ring and is solvent accessible.Moreover, the entry of this pocket is located in close proximity of theactive site (D251, D343 and D344) of the enzyme, which is absolutelyessential for telomerase function. Alanine mutants of any of the abovethree invariant aspartates that form the active site of the enzymecompletely abolishes telomerase activity.

Based on the fact that the FP-pocket is a stable, well-defined, solventaccessible cavity and is located in close proximity of the active siteof the enzyme indicates that it is likely a useful target for theidentification of small molecule inhibitors-activators that modulatetelomerase activity. Based on the structure of the FP-pocket, it isexpected that inhibitors will occlude the active site of telomerase thusinterfering with nucleotide association and therefore telomerereplication. On the other hand, activators are expected to be in closeproximity to the active site of the enzyme without occluding the activesite of the enzyme. These compounds could therefore be involved infavorable interactions with the incoming nucleotide substrate producinga tight and stable catalytic active complex thus increasing telomeraseactivity.

Example 5 Modulators of the T-Pocket

Based upon the structure of the T-pocket, an in silico screen wascarried out to identify small molecules that associate or interact withthis pocket. Compounds were screened for their ability to disrupt theproper association of the catalytic subunit of telomerase TERT with theRNA component TER that binds in this location, thereby modulatingtelomerase function. Structurally similar groups of compounds,identified via in silico screening as making extensive contacts withmotifs T, 1 and 2 of the T-pocket, are listed in Table 8.

TABLE 8 Group Compound 1 6-[bis(phosphonomethyl)amino]hexanoic acid 2(2-{2-[(6-oxo-1,6-dihydro-3-pyridazinyl)carbonyl]carbonohydrazonoyl}phenoxy)aceticAcid 3,5-dihydroxy-N′-(3-hydroxy-4-methoxybenzylidene)benzohydrazide2-hydroxy-N′-(3-hydroxy-4-methoxybenzylidene)benzohydrazideN′-(3-bromo-4-hydroxy-5-methoxybenzylidene)-3,4-dihydroxybenzohydrazide4-(2-{1-[3-(aminocarbonyl)-4-hydroxyphenyl]ethylidene}hydrazino)benzoicacid 4-(2-{oxo[(1-phenylethyl)amino]acetyl}carbonohydrazonoyl)benzoicacid 3-hydroxy-N′-(4-hydroxy-3,5-dimethoxybenzylidene)benzohydrazideN′-[1-(2,4-dihydroxyphenyl)ethylidene]-4-(2-oxo-1-pyrrolidinyl)benzohydrazide 3 5-{[(4-methoxyphenyl)acetyl]amino}isophthalic acidphenyl 2-hydroxy-4-[(phenoxyacetyl)amino]benzoate4,4′-[iminobis(methylene)]dibenzoic acid2,2′-[1,3-phenylenebis(carbonylimino)]bis(5-hydroxybenzoic acid)3-{[3-(2-furoylamino)benzoyl]amino}-2-methylbenzoic acid phenyl3-({[4-(propionylamino)benzoyl]oxy}methyl)benzoate4-{[(8-allyl-2-oxo-2H-chromen-3-yl)carbonyl]amino}benzoic acid2,5-bis[(phenylacetyl)amino]terephthalic acid5-{[(2-naphthyloxy)acetyl]amino}isophthalic acid4-[({3-[(tetrahydro-2-furanylcarbonyl)amino]phenyl}amino)carbonyl]benzoicacid 5-{[(3,4-dimethylphenoxy)acetyl]amino}-2-hydroxybenzoic acid(4-{[4-(2-furoylamino)benzoyl]amino}phenyl)acetic acid3,4-dimethyl-6-{[(4-{[(3-pyridinylmethyl)amino]carbonyl}phenyl)amino]carbonyl}-3-cyclohexene-1-carboxylic acid6-({[4-(methoxycarbonyl)phenyl]amino}carbonyl)-3,4-dimethyl-3-cyclohexene-1-carboxylic acidN-[4-(acetylamino)phenyl]-8-allyl-2-oxo-2H-chromene-3-carboxamide5-bromo-2-[(4-{[(phenylthio)acetyl]amino}benzoyl)amino]benzoic acidN-(4-acetylphenyl)-8-allyl-2-oxo-2H-chromene-3-carboxamideN-[4-(acetylamino)phenyl]-N′-benzylethanediamide5-{[(3-methoxyphenoxy)acetyl]amino}isophthalic acid2-({3-[(4-carboxybutanoyl)amino]benzoyl}amino)benzoic acid6-({[4-(ethoxycarbonyl)phenyl]amino}carbonyl)-3,4-dimethyl-3-cyclohexene-1-carboxylic acid1-(4-{[(4-chlorophenyl)acetyl]amino}phenyl)-N-(4-methoxyphenyl)cyclopentanecarboxamide3-({[(4-sec-butoxybenzoyl)amino]carbonothioyl}amino)benzoic acid]N,N′-di-2-naphthylterephthalamide ethyl4-{[(5-bromo-4-formyl-2-methoxyphenoxy)acetyl]amino}benzoate4,4′-[1,4-phenylenebis(carbonylimino)]dibenzoic acid 4N-(3-hydroxyphenyl)-3-phenylacrylamide3-(2-chlorophenyl)-N-(3-hydroxyphenyl)acrylamide2-bromo-N-{4-[3-(4-methoxyphenyl)acryloyl]phenyl}benzamideN-[1-{[(4-acetylphenyl)amino]carbonyl}-2-(1,3-benzodioxol-5-yl)vinyl]-2-Chlorobenzamide4-(4-hydroxy-3-methoxyphenyl)-N-(4-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydro-5-pyrimidinecarboxamide 5N-(2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl)-2-(3-methoxyphenyl)-1,3-dioxo-5-isoindolinecarboxamide5-bromo-2-{[(2-cyclohexyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)carbonyl]amino}benzoic acid5-{[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzoyl]amino}isophthalicacid 5-({[2-(2-methoxyethyl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]carbonyl}amino)isophthalic acid 63-hydroxy-1-(1-naphthylmethyl)-3-[2-oxo-2-(3-pyridinyl)ethyl]-1,3-dihydro-2H-indol-2-oneN-(4-methoxyphenyl)-2-{[1-(2-methylphenyl)-2,5-dioxo-3-pyrrolidinyl]thio}acetamide3-hydroxy-3-[2-(4-methoxyphenyl)-2-oxoethyl]-1-(4-morpholinylmethyl)-1,3-dihydro-2H-indol-2-one5-chloro-1-(2-chlorobenzyl)-3-hydroxy-3-[2-oxo-2-(4-pyridinyl)ethyl]-1,3-dihydro-2Hindol-2-one3-hydroxy-3-[2-(4-methoxyphenyl)-2-oxoethyl]-1-propyl-1,3-dihydro-2H-indol-2-one3-[2-(1,3-benzodioxol-5-yl)-2-oxoethyl]-5-chloro-3-hydroxy-1-(4-methylbenzyl)-1,3-dihydro-2H-indol-2-one3-hydroxy-3-[2-(4-methoxyphenyl)-2-oxoethyl]-1-methyl-1,3-dihydro-2H-indol-2-one3-[2-(1,3-benzodioxol-5-yl)-2-oxoethyl]-3-hydroxy-1,3-dihydro-2H-indol-2-one3-[2-(1,3-benzodioxol-5-yl)-2-oxoethyl]-3-hydroxy-1-(3-phenyl-2-propen-1-yl)-1,3-dihydro-2H-indol-2-one5-bromo-3-hydroxy-3-[2-(4-methoxyphenyl)-2-oxoethyl]-1-methyl-1,3-dihydro-2H-indol-2-oneN-(4-methoxyphenyl)-2-{[1-(2-methylphenyl)-2,5-dioxo-3-pyrrolidinyl]thio}acetamide3-[2-(1,3-benzodioxol-5-yl)-2-oxoethyl]-3-hydroxy-6,7-dimethyl-1,3-dihydro-2H-indol-2-one 75-{[(3-amino-4-chlorophenyl)sulfonyl]amino}isophthalic acid3-[(3-{[(4-carboxyphenyl)amino]sulfonyl}benzoyl)amino]benzoic acid 82-([1]benzofuro[3,2-d]pyrimidin-4-ylthio)-N-(3- hydroxyphenyl)acetamideN-(3-hydroxyphenyl)-2-[(6-methylthieno[2,3-d]pyrimidin-4-yl)thio]acetamide 2-{[7-(2-furylmethyl)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]thio}-N-(3-hydroxyphenyl)acetamideN-[4-(1,3-benzothiazol-2-ylmethoxy)-2-methylphenyl]-2-(3-methylphenoxy)acetamide2-{[5-(2-hydroxyethyl)-4-methyl-6-oxo-1,6-dihydro-2-pyrimidinyl]thio}-N-(3-methoxyphenyl)acetamideN-(3-acetylphenyl)-2-[(5,6-dimethylthieno[2,3-d]pyrimidin-4-yl)thio]acetamide 9N-({5-[(2-amino-2-oxoethyl)thio]-4-methyl-4H-1,2,4-triazol-3-yl}methyl)-2-(4-methoxyphenyl)acetamideN-(3-hydroxyphenyl)-2-{[4-methyl-5-(1-phenoxyethyl)-4H-1,2,4-triazol-3-yl]thio}acetamide2,4-dichloro-N-{1-[5-({2-[(4-methoxyphenyl)amino]-2-oxoethyl}thio)-4-methyl-4H-1,2,4-triazol-3- yl]ethyl}benzamide 10 methyl2-{[({1-[4-(methoxycarbonyl)phenyl]-1H-tetrazol-5-yl}thio)acetyl]amino}benzoate 112′-{[(6-methoxy-1,3-benzothiazol-2-yl)amino]carbonyl}-2-biphenylcarboxylic acid2-{[4-amino-5-(phenoxymethyl)-4H-1,2,4-triazol-3-yl]thio}-N-1,3-benzothiazol-2-ylacetamide2-[(5-oxo-4-phenyl-4,5-dihydro-1H-1,2,4-triazol-3-yl)thio]-N-1,3-thiazol-2-ylpropanamide2′-{[(6-ethoxy-1,3-benzothiazol-2-yl)amino]carbonyl}-2-biphenylcarboxylic acid 124-[(4-{[3-(4-chlorophenyl)-2,4-dioxo-1,3-thiazolidin-5-ylidene]methyl}phenoxy)methyl]benzoic acid3-[5-(4-ethoxybenzylidene)-4-oxo-2-thioxo-1,3- thiazolidin-3-yl]benzoicacid methyl 4-({4-oxo-3-[4-oxo-4-(1,3-thiazol-2-ylamino)butyl]-2-thioxo-1,3-thiazolidin-5-ylidene}methyl)benzoate 134-[5-[4-(allyloxy)-3-chloro-5-methoxybenzylidene]-2,4,6-trioxotetrahydro-1(2H)-pyrimidinyl]benzoic acid methyl4-[5-(3-bromo-5-ethoxy-4-hydroxybenzylidene)-2,4,6-trioxotetrahydro-1(2H)-pyrimidinyl]benzoate4-[5-(4-hydroxy-3-methoxybenzylidene)-2,4,6-trioxotetrahydro-1(2H)-pyrimidinyl]benzoic acid2-chloro-4-(5-{[1-(2-fluorobenzyl)-2,5-dioxo-4-imidazolidinylidene]methyl}-2-furyl)benzoic acid methyl4-[5-(4-hydroxy-3-methoxybenzylidene)-2,4,6-trioxotetrahydro-1(2H)-pyrimidinyl]benzoate4-({4-[(1,3-dimethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]phenoxy}methyl)benzoic acid4-[5-[4-(allyloxy)-3-methoxybenzylidene]-2,4,6-trioxotetrahydro-1(2H)-pyrimidinyl]benzoic acid4-({4-[(1-ethyl-5-oxo-2-thioxo-4-imidazolidinylidene)methyl]phenoxy}methyl)benzoic acid4-[5-[3-methoxy-4-(2-propyn-1-yloxy)benzylidene]-2,4,6-trioxotetrahydro-1(2H)-pyrimidinyl]benzoic acid4-[5-(3-methoxy-4-propoxybenzylidene)-2,4,6-trioxotetrahydro-1(2H)-pyrimidinyl]benzoic acid4-(5-{[1-(2-fluorobenzyl)-2,5-dioxo-4-imidazolidinylidene]methyl}-2-furyl)benzoic acid4-[5-[3-bromo-5-methoxy-4-(2-propyn-1-yloxy)benzylidene]-2,4,6-trioxotetrahydro-1(2H)-pyrimidinyl]benzoic acid methyl4-[5-(3-methoxybenzylidene)-2,4,6-trioxotetrahydro-1(2H)-pyrimidinyl]benzoate 143-[(5-{[5-(2,5-dichlorophenyl)-2-furyl]methylene}-4-oxo-1,3-thiazolidin-2-ylidene)amino]benzoic acid3-{5-[(2-imino-4-oxo-1,3-thiazolidin-5-ylidene)methyl]-2- furyl}benzoicacid 3-(5-{[5-imino-2-(2-methylphenyl)-7-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-6(7H)-ylidene]methyl}-2-furyl)benzoic acid 155-({[2-(4-methoxyphenyl)ethyl]amino}methylene)-2,4,6(1H,3H,5H)-pyrimidinetrione 165-{[1-(2-chlorophenyl)-2,5-dioxo-3-pyrrolidinyl]amino}-2,4(1H,3H)-pyrimidinedione 17 4-formylphenyl(2,4-dioxo-1,3-thiazolidin-5-yl)acetate 4-formyl-2-methoxyphenyl(2,4-dioxo-1,3-thiazolidin-5-yl)acetate 18 disodium7-hydroxy-6-sulfo-1,3-naphthalenedisulfonate disodium4-hydroxy-5-sulfo-2,7-naphthalenedisulfonate sodium4-amino-5-sulfo-2-naphthalenesulfonate 192,2′-{[(4-methoxyphenyl)sulfonyl]imino}diacetic acid

These compounds, as well as derivatives and analogs thereof, areexpected to modulate the activity of telomerase. Accordingly, thepresent invention relates to effector compounds selected for interactingwith motifs T, and 2 of the T-pocket of telomerase. In particularembodiments, a compound of the invention is selected from the grouplisted in Table 8. In another embodiment, one or more of the compoundslisted in Table 8 serve as lead compounds for designing or generatingderivatives or analogs which are more potent, more specific, less toxicand more effective than known inhibitors of telomerase or the leadcompound. Derivatives or analogs can also be less potent but have alonger half-life in vivo and/or in vitro and therefore be more effectiveat modulating telomerase activity in vivo and/or in vitro for prolongedperiods of time.

Derivatives or analogs of the compounds disclosed herein typicallycontain the essential elements of the parent compound, but have had oneor more atoms (e.g., halo, lower alkyl, hydroxyl, amino, thiol, ornitro), or group of atoms (e.g., amide, aryl, heteroaryl, allyl, orpropargyl), replaced or added. Such replacements or substitutions caninclude substituent R groups and/or atoms of the core structure, e.g.,replacing a carbon with a heteroatom such as a nitrogen, oxygen, orsulfur. In this regard, the compounds disclosed herein serve as leadcompounds for creating a family of derivatives or analogs of use ininhibiting or activating telomerase to, e.g., alter lifespan orproliferative capacity of a cell.

Example 6 Modulators of the FP-Pocket of Telomerase

X-ray crystal structure indicates that the organization of the fingersand palm domains between the substrate-free and substrate-bound TERTmolecules is highly similar, indicating that like the Hepatitis C viralRNA polymerase (NS5B), telomerase has a preformed active site. Thisobservation is surprising since in most polymerases, including the HIVreverse transcriptase, the fingers domain undergoes significantconformational changes referred to as the open and closed states. In HIVreverse transcriptase, conformational rearrangements of the fingersdomain with respect to the palm domain are essential for nucleotidebinding and positioning at the active site of the enzyme and theirabsence from telomerase suggests possible mechanistic differences innucleotide binding and selectivity between these families of enzymes.

The organization of the fingers and palm domains of TERT creates a deep,well-defined, narrow, mostly hydrophobic Fingers-Palm pocket(FP-pocket). The FP-pocket, generated by residues Y170, F193, A195,D254, W302, H304, L307, Q308 (with reference to Tribolium castaneumtelomerase; FIG. 8), opens into the interior cavity of the TERT ring andis solvent accessible. Moreover, the entry of this pocket is located inclose proximity of the active site (D251, D343 and D344 of T. castaneumtelomerase) of the enzyme, which is absolutely essential for telomerasefunction; alanine mutants of any of the three invariant aspartatescompletely abolishes telomerase activity. In so far as the FP-pocket isa stable, well-defined, solvent accessible cavity and is located inclose proximity of the active site of the enzyme, this pocket wasselected as a target for the identification of effector molecules thatmodulate telomerase activity.

Accordingly, based upon the structure of the FP-pocket, an in silicoscreen was carried out to identify small molecules that associate orinteract with this pocket (FIG. 9). Inhibitors of telomerase wereselected for their ability to occlude the active site of telomerase thusinterfering with nucleotide association and therefore telomerereplication. On the other hand, activators were selected for being inclose proximity to the active site of the enzyme without occluding theactive site. Activators could therefore be involved in favorableinteractions with the incoming nucleotide substrate producing a tightand stable catalytic active complex thus increasing telomerase activity.Structurally similar groups of compounds, identified via in silicoscreening as making extensive contacts with the FP-pocket, are listed inTable 9.

TABLE 9 Group Compound 12-(acetylamino)-N-(aminocarbonyl)-3-thiophenecarboxamideN-(3-{[(aminocarbonyl)amino]carbonyl}-2- thienyl)isonicotinamideN-(aminocarbonyl)-2-{[(5-methyl-3-thienyl)carbonyl]amino}-3-thiophenecarboxamideN-(3-{[(aminocarbonyl)amino]carbonyl}-2-thienyl)-2-oxo-2H-chromene-3-carboxamide ethyl2-[(4-amino-4-oxo-2-butenoyl)amino]-4,5-dimethyl-3- thiophenecarboxylate2-(benzoylamino)-N-(3-hydroxypropyl)-4,5-dimethyl-3-thiophenecarboxamide 6-tert-butyl-2-[(2-fluorobenzoyl)amino]-N-(2-hydroxyethyl)-4,5,6,7-tetrahydro-1-benzothiophene-3- carboxamide 2 ethyl5-cyano-6-{[(5-hydroxy-1H-pyrazol-3-yl)methyl]thio}-2-methyl-4-(2-thienyl)-1,4-dihydro-3-pyridinecarboxylate 32-[(5-acetyl-3-cyano-6-methyl-2-pyridinyl)thio]acetamide 43-amino-N-(3-hydroxypropyl)-7,7-dimethyl-7,8-dihydro-5H-thieno[2,3-b]thiopyrano[3,4-e]pyridine-2-carboxamide 51,4-benzothiazin-2-yl)acetamide1,2,3,4-tetrahydro-2-quinoxalinyl)acetamideN-(4-fluorophenyl)succinamide 6 N-(3-methylphenyl)dicarbonimidic diamideN-(2-methoxyphenyl)dicarbonimidic diamideN-(4-chlorophenyl)dicarbonimidic diamideN-(4-methoxyphenyl)dicarbonimidic diamide 7N-(2-hydroxyethyl)-N′-(2-methoxyphenyl)ethanediamideN-(3-fluorophenyl)-N′-(2-hydroxyethyl)ethanediamideN-(2-hydroxyethyl)-N′-(2-methylphenyl)ethanediamideN-1,3-benzodioxol-5-yl-N′-(2-hydroxypropyl)ethanediamideN-(5-chloro-2-methylphenyl)-N′-(3- hydroxypropyl)ethanediamide3,5-dichloro-2-[(2,4-dichlorobenzoyl)amino]-N-(2- hydroxyethyl)benzamide8 1-amino-3-[(2-chloro-4-nitrophenyl)amino]-2-propanol 93-bromo-N-(1-{[(2-hydroxyethyl)amino]carbonyl}-2-(3-nitrophenyl)vinyl]benzamide N-(2-(2-fluorophenyl)-1-{[(3-hydroxypropyl)amino]carbonyl}vinyl)benzamide3-bromo-N-(2-(2-furyl)-1-{[(3-hydroxypropyl)amino]carbonyl}vinyl)benzamide 10 N-2~-acetylarginine 111-[(4-ethoxy-3-methylphenyl)sulfonyl]-N-(2-hydroxyethyl)-3-piperidinecarboxamide N-(3-hydroxypropyl)-1-(2-naphthylsulfonyl)-3-piperidinecarboxamide 12N-[amino(imino)methyl]-4-{[3-(1,3-benzodioxol-5-yl)-3-oxo-1-propen-1-yl]amino}benzenesulfonamide 133-(3-{[(3-hydroxypropyl)amino]sulfonyl}-4,5- dimethoxyphenyl)acrylicacid 14 2-[2-chloro-4-({[2-(2-fluorophenyl)ethyl]amino}methyl)-6-methoxyphenoxy]acetamide 2-(2-chloro-6-methoxy-4-{[(4-pyridinylmethyl)amino]methyl} phenoxy)acetamidehydrochloride2-(2-chloro-6-methoxy-4-{[(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)amino]methyl}phenoxy)acetamide2-{2-chloro-6-methoxy-4- [(methylamino)methyl]phenoxy}acetamidehydrochloride 2-(2-chloro-6-methoxy-4-{[(3-pyridinylmethyl)amino]methyl}phenoxy) acetamidehydrochloride2-{4-[(1-adamantylamino)methyl]-2-chloro-6- methoxyphenoxy}acetamide2-(2-chloro-6-ethoxy-4-{[(3- pyridinylmethyl)amino]methyl}phenoxy)acetamide hydrochloride 2-{2-chloro-4-[(cyclooctylamino)methyl]-6-methoxyphenoxy}acetamide hydrochloride 2-(2-chloro-6-ethoxy-4-{[(2-thienylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-(2-bromo-6-methoxy-4-{[(3-pyridinylmethyl)amino]methyl}phenoxy)acetamide hydrochloride 2-(2-chloro-6-methoxy-4-{[(2-thienylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-(2-chloro-4-{[(2-hydroxypropyl)amino]methyl}-6- methoxyphenoxy)acetamide 2-[2-bromo-6-methoxy-4-({[2-(4-morpholinyl)ethyl]amino}methyl) phenoxy]acetamide dihydrochloride2-{4-[(2-adamantylamino)methyl]-2-chloro-6- methoxyphenoxy}acetamide2-(2-bromo-6-methoxy-4-{[(4-pyridinylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-(2-chloro-6-ethoxy-4-formylphenoxy)acetamide2-(2-chloro-6-methoxy-4-{[(tetrahydro-2- furanylmethyl)amino]methyl}phenoxy)acetamide hydrochloride 2-{4-[(benzylamino)methyl]-2-chloro-6-methoxyphenoxy}acetamide hydrochloride2-{2-chloro-4-[(cyclopentylamino)methyl]-6- methoxyphenoxy}acetamidehydrochloride 2-[2-chloro-4-(hydroxymethyl)-6-methoxyphenoxy]acetamide2-(2-chloro-4-{[(4-fluorobenzyl)amino]methyl}-6-methoxyphenoxy)acetamide hydrochloride2-{2-chloro-4-[(cyclopropylamino)methyl]-6- ethoxyphenoxy}acetamidehydrochloride 2-(2-bromo-6-methoxy-4-{[(2-phenylethyl)amino]methyl}phenoxy)acetamide2-{2-chloro-4-[(cyclopropylamino)methyl]-6- methoxyphenoxy}acetamidehydrochloride 2-(2-chloro-6-ethoxy-4-{[(2-furylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-[2-chloro-4-({[2-(4-fluorophenyl)ethyl]amino}methyl)-6-methoxyphenoxy]acetamide 2-(2-bromo-6-ethoxy-4-{[(2-furylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-(2-bromo-6-methoxy-4-{[(2-thienylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-{2-chloro-6-ethoxy-4- [(isopropylamino)methyl]phenoxy}acetamide2-[2-bromo-6-ethoxy-4-(hydroxymethyl)phenoxy]acetamide2-{2-chloro-4-[(cyclohexylamino)methyl]-6- methoxyphenoxy}acetamidehydrochloride 2-(2-bromo-6-methoxy-4-{[(tetrahydro-2-furanylmethyl)amino]methyl} phenoxy)acetamide hydrochloride2-{4-[(butylamino)methyl]-2-chloro-6- methoxyphenoxy}acetamide 152-(4-bromophenoxy)ethanimidamide hydrochloride 162-(1,3-benzodioxol-5-ylmethylene)hydrazinecarboximidamide2-(4-methoxybenzylidene)hydrazinecarboximidamide3-allyl-2-hydroxybenzaldehyde semicarbazone 17N-[(4-methoxybenzyl)oxy]guanidine sulfate 182-(8-quinolinyl)hydrazinecarboxamide 19N-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-1H-pyrazole-5-carboxamide 20 7-methyl-1H-indole-2,3-dione 3-semicarbazone21 N-(aminocarbonyl)-3-oxo-3,4-dihydro-2- quinoxalinecarboxamide 221-benzyl-N-(3-hydroxypropyl)-4-oxo-1,4-dihydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-2-carboxamide 232-{[7-(2-hydroxy-3-phenoxypropyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl]thio}acetamide 246-[(4-bromo-2,3-dimethylphenyl)amino]-2-(3-hydroxypropyl)-5-nitro-1Hbenzo[de]isoquinoline- 1,3(2H)-dione3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propanamide 252-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-4H-1,2,4-triazol-4-ylpropanamide 26 N-[amino(imino)methyl]-3,4-dihydro-1(2H)-quinolinecarboximidamide 27 N-(4,6-dimethyl-2-quinazolinyl)guanidineN-(4,6,7-trimethyl-2-quinazolinyl)guanidineN-(4,6,8-trimethyl-2-quinazolinyl)guanidineN-(4,8-dimethyl-2-quinazolinyl)guanidineN-(4,7-dimethyl-2-quinazolinyl)guanidineN-(4-methyl-2-quinazolinyl)guanidineN-(4-methoxy-6-methyl-2-quinazolinyl)guanidine 28N-1,3-benzoxazol-2-ylguanidine 293-amino-N′-(2,4-dichlorobenzylidene)-1H-1,2,4-triazole-5- carbohydrazide30 3-chloro-4-fluoro-N-4H-1,2,4-triazol-4-yl-1-benzothiophene-2-carboxamide5-bromo-2-chloro-N-4H-1,2,4-triazol-4-ylbenzamide2,5-dichloro-N-4H-1,2,4-triazol-4-ylbenzamide oxalate2-chloro-4-methyl-N-4H-1,2,4-triazol-4-ylbenzamide2,4-dimethyl-N-4H-1,2,4-triazol-4-ylbenzamide2,3-dichloro-N-4H-1,2,4-triazol-4-ylbenzamide 31N-[amino(imino)methyl]-4-morpholinecarboximidamide hydrochloride

To demonstrate activity, selected compounds from Table 9 were analyzedfor inhibitory activity. The results of this analysis are presented inTable 10.

TABLE 10 Inhibition Inhibition at 50 μM at 5 μM Compound Structure/Name(%)* (%)*

49.10 17.68 2-(2-chloro-6-ethoxy-4-{[(2-thienylmethyl)amino]methyl}phenoxy) acetamide hydrochloride

45.56 23.05 2-(2-chloro-6-methoxy-4-{[(4-pyridinylmethyl)amino]methyl}phenoxy) acetamide hydrochloride

41.26 10.94 2-(2-chloro-6-methoxy-4-{[(2-thienylmethyl)amino]methyl}phenoxy) acetamide hydrochloride

39.41 19.23 ethyl 2-[(4-amino-4-oxo-2- butenoyl)amino]-4,5-dimethyl-3-thiophenecarboxylate

38.78 14.87 2-(2-chloro-6-ethoxy-4-{[(3-pyridinylmethyl)amino]methyl}phenoxy) acetamide hydrochloride

36.65  8.14 3-bromo-N-[1-{[(2- hydroxyethyl)amino]carbonyl}-2-(3-nitrophenyl)vinyl]benzamide

36.58 18.11 3-(1,3-dioxo-1H-benzo[de]isoquinolin- 2(3H)-yl)propanamide

35.18 13.73 2-{4-[(2-adamantylamino)methyl]-2-chloro-6-methoxyphenoxy}acetamide

31.30 17.00 2-{2-chloro-6-methoxy-4- [(methylamino)methyl]phenoxy}acetamide hydrochloride

31.01 11.80 3-bromo-N-(2-(2-furyl)-1-{[(3-hydroxypropyl)amino]carbonyl}vinyl) benzamide

30.92 16.14 2-(2-bromo-6-ethoxy-4-{[(2-furylmethyl)amino]methyl}phenoxy) acetamide hydrochloride

30.42 13.44 N-(3-hydroxypropyl)-1-(2- naphthylsulfonyl)-3-piperidinecarboxamide

30.23 18.27 2-[2-bromo-6-methoxy-4-({[2-(4-morpholinyl)ethyl]amino}methyl) phenoxy]acetamide dihydrochloride

30.04 11.97 5-bromo-2-chloro-N-4H-1,2,4-triazol-4- ylbenzamide

29.96  4.96 2-{4-[(benzylamino)methyl]-2-chloro-6-methoxyphenoxy}acetamide hydrochloride

29.71  6.67 N-1,3-benzodioxol-5-yl-N′-(2- hydroxypropyl)ethanediamide

29.35 10.94 2-chloro-4-methyl-N-4H-1,2,4-triazol- 4-ylbenzamide

29.33 15.94 2-(2-chloro-6-methoxy-4-{[(2-oxo-2,3-dihydro-1H-benzimidazol-5- yl)amino]methyl}phenoxy)acetamide

27.65 12.21 2-(2-bromo-6-methoxy-4-{[(2-phenylethyl)amino]methyl}phenoxy) acetamide

27.38  4.74 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-4H-1,2,4-triazol-4- ylpropanamide *The observed inhibitionconstant corresponds to 50% of the actual inhibition value.Therefore, ifthe observed value is 49% the actual value is 98%.

In one embodiment, compounds of the present invention have the structureof Formula I:

wherein R₁ is —H or —CH₃; R₂ is a substituent as found in the compoundsof Tables 9 or 10; and R₃ is a halo group (I, F, Br, Cl).

In another embodiment, compounds of the present invention have thestructure of Formula II:

wherein R₄ is a halo group (I, F, Br, Cl); and R₅ is a substituent asfound in the compounds of Tables 9 or 10.

As demonstrated by the data presented in Table 10, compounds of Table 9,as well as derivatives and analogs thereof (including derivatives andanalogs of Formula I and Formula II), are expected to modulate theactivity of telomerase. Accordingly, the present invention relates toeffector compounds selected for interacting with the FP-pocket oftelomerase. In particular embodiments, a compound of the invention isselected from the group listed in Table 9. In another embodiment, one ormore of the compounds listed in Table 9 serve as lead compounds fordesigning or generating derivatives or analogs which are more potent,more specific, less toxic and more effective than known inhibitors oftelomerase or the lead compound. Derivatives or analogs can also be lesspotent but have a longer half-life in vivo and/or in vitro and thereforebe more effective at modulating telomerase activity in vivo and/or invitro for prolonged periods of time.

Derivatives or analogs of the compounds disclosed herein typicallycontain the essential elements of the parent compound, but have had oneor more atoms (e.g., halo, lower alkyl, hydroxyl, amino, thiol, ornitro), or group of atoms (e.g., amide, aryl, heteroaryl, allyl, orpropargyl), replaced or added. Such replacements or substitutions caninclude substituent R groups and/or atoms of the core structure, e.g.,replacing a carbon with a heteroatom such as a nitrogen, oxygen, orsulfur. In this regard, the compounds disclosed herein serve as leadcompounds for creating a family of derivatives or analogs of use ininhibiting or activating telomerase to, e.g., alter lifespan orproliferative capacity of a cell.

Example 7 Fluorescence Polarization Binding Assay

The TERT protein was expressed and purified as described herein. Proteinwas concentrated to 4.72 mg ml-l using an AMICON 30K cutoff filter(Millipore) and stored at −80° C. for binding assays.

For the binding assay, TERT protein was diluted to a 500 nMconcentration in a solution of 50 mM Tris-HCl, pH 7.5, 200 mM KCl, and20% glycerol. A 1.2-fold molar excess of the Chim21m RNA-DNA hybrid(Integrated DNA Technologies), containing the template for telomereextension (5′-rCrUrG rArCrC rUrGrA rCTT CGG TCA GGT-3′; SEQ ID NO:19),was then added to these samples and kept on ice. The cyanine 5-dCTP(Perkin Elmer) for the binding study was made up to a 20 nMconcentration in sterile, RNase, DNase free water with 40 nM MgCl₂.

Compound screens were carried out as 30.5 μl reactions in a 384-well,round bottom plate (Thermo). Fifteen μl of TERT-Chim21m complex wasfirst added to each well. Then 0.5 μl of library compound (ChemBridge)was added to this solution. Finally, 15 μl of the nucleotide-MgCl2sample was added, and the solution mixed to provide final concentrationsof 250 nM protein-nucleic acid, 10 nM cyanine 5-dCTP, and 20 nM MgCl₂.Final compound concentrations were 50 μM and 5 μM. All additions werecompleted using a Janus 96/384 Modular Dispensing Tool (Perkin Elmer),performed in triplicate, and equilibrated for 40 minutes. Fluorescencepolarization was measured at λ_(ex)=620 nm and λ_(em)=688 nm on anEnVision Xcite Multilabel plate reader (Perkin Elmer). Polarization wascalculated using the standard equation: P=(V−H)/(V+H), where P denotespolarization, V denotes vertical emission intensity, and H denoteshorizontal emission intensity.

Example 8 Activity of Telomerase Inhibitors Against Breast Cancer Cellsand Cell Lysate

Compounds were screened for their ability to inhibit the telomeraseactivity in breast cancer cells (MCF-7) and cell lysates. Compoundshaving IC₅₀ values in the range of 0.168 μM and 8.48 μM are listed inTable 11.

TABLE 11 Group Molecular Name 1N-(2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl)-2-(4-nitrophenyl)-1,3-dioxo-5-isoindolinecarboxamideN-(4-nitrophenyl)-2-(4-{[(4-nitrophenyl)amino]carbonyl}phenyl)-1,3-dioxo-5- isoindolinecarboxamideN-(2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl)-2-(3-nitrophenyl)-1,3-dioxo-5-isoindolinecarboxamideN-(4-hydroxyphenyl)-2-(4-{[(4-hydroxyphenyl)amino]carbonyl}phenyl)-1,3-dioxo-5- isoindolinecarboxamideN-(3-acetylphenyl)-2-(4-{[(3-acetylphenyl)amino]carbonyl}phenyl)-1,3-dioxo-5- isoindolinecarboxamideN-(4-bromophenyl)-2-(4-{[(4-bromophenyl)amino]carbonyl}phenyl)-1,3-dioxo-5- isoindolinecarboxamideN-(2-hydroxy-5-methylphenyl)-2-(4-{[(2-hydroxy-5-methylphenyl)amino]carbonyl}phenyl)-1,3-dioxo-5- isoindolinecarboxamideN-(4-chlorophenyl)-2-(4-{[(4-chlorophenyl)amino]carbonyl}phenyl)-1,3-dioxo-5- isoindolinecarboxamideN-(2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl)-2-(4-{[(2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl)amino]carbonyl}phenyl)-1,3-dioxo-5- isoindolinecarboxamide 22-{[3-(4-{[(2- carboxyphenyl)amino]carbonyl}phenoxy)benzoyl]amino}-benzoic acid 2,2′-[[5-(benzoylamino)-1,3-phenylene]bis(carbonylimino)]dibenzoic acid3,3′-methylenebis{6-[(2-thienylacetyl)amino]benzoic acid}3-[(2-{[1-(4-fluorobenzyl)-2,5-dioxo-4- imidazolidinylidene]methyl}-6-methoxyphenoxy)methyl]benzoic acid3,3′-methylenebis[6-(benzoylamino)benzoic acid] 34,4′-bis{[4-(acetyloxy)benzoyl]amino}-3,3′- biphenyldicarboxylic acid 42,2′-[1,2,4-thiadiazole-3,5-diylbis(thio)]bis{N-[4-(aminosulfonyl)phenyl]acetamide}

Given their activity, the present invention further provides thecompounds listed in Table 11, as well as pharmaceutical salts andpharmaceutical compositions thereof, for use in inhibiting the activityof telomerase and in the treatment of the diseases and conditions listedherein. In another embodiment, one or more of the compounds listed inTable 11 serve as lead compounds for designing or generating derivativesor analogs which are more potent, more specific, less toxic and moreeffective than known inhibitors of telomerase or the lead compound.Derivatives or analogs can also be less potent but have a longerhalf-life in vivo and/or in vitro and therefore be more effective atmodulating telomerase activity in vivo and/or in vitro for prolongedperiods of time.

Derivatives or analogs of the compounds disclosed in Table 11 typicallycontain the essential elements of the parent compound, but have had oneor more atoms (e.g., halo, lower alkyl, hydroxyl, amino, thiol, ornitro), or group of atoms (e.g., amide, aryl, heteroaryl, allyl, orpropargyl), replaced or added. Such replacements or substitutions caninclude substituent R groups and/or atoms of the core structure, e.g.,replacing a carbon with a heteroatom such as a nitrogen, oxygen, orsulfur. In this regard, the compounds disclosed herein serve as leadcompounds for creating a family of derivatives or analogs of use ininhibiting or activating telomerase to, e.g., alter lifespan orproliferative capacity of a cell.

Accordingly, in one embodiment, compounds of the present invention havethe structure of Formula III:

wherein R₆ is a substituted or unsubstituted benzene or heterocyclicgroup and R₇ is —NO₂ or —C(O)NH—R₈, wherein R₈ is a substituted orunsubstituted benzene or heterocyclic group. When substituted, thebenzene or heterocyclic groups of Formula III can contain between 1 and4 substituents at the ortho, para or meta positions. Substituentsinclude, but are not limited to lower alkyl (C1-C4, e.g., methyl orethyl groups), amine (—NH₂), nitro (—NO₂), oxygen (═O), hydroxyl (—OH),C(O)CH₃, or halo (Br, Cl, I, or F) groups. Heterocyclic groups of theinvention include C5 and C6 heterocyles such as imidazolidones,hydantoins, pyrazoles, imidazoles, pyridines, and pyrimidines. Exemplarycompounds and substituents of Formula III are provided in Table 11.

In another embodiment, compounds of the present invention have thestructure of Formula IV:

wherein R₉ and its dashed bond are either present or absent; R₁₀ and R₁₁are independently hydrogen (—H) or carboxyl (—COOH) groups; and R₁₂ andR₁₃ are independently —C(O)NH— or —NHC(O)—; R₁₄ and R₁₅ areindependently substituted or unsubstituted benzene or heterocyclicgroups. When present, R₉ is oxygen (O) or carbon (C). When substituted,the benzene or heterocyclic groups of Formula IV can contain between 1and 4 substituents at the ortho, para or meta positions. Substituentsinclude, but are not limited to lower alkyl (C₁-C₄, e.g., methyl orethyl groups), amine (—NH₂), nitro (—NO₂), oxygen (═O), hydroxyl (—OH),carboxyl (—COOH), C(O)CH₃, —OC(O)CH₃, or halo (Br, Cl, I, or F) groups.Heterocyclic groups of the invention include C5 and C6 heterocyles suchas imidazolidones, hydantoins, pyrazoles, imidazoles, thiophenes,pyridines, and pyrimidines. Exemplary compounds and substituents ofFormula IV are provided in Table 11.

Example 9 Efficacy of Telomerase Inhibitors

Novel telomerase inhibitors of the instant invention can be analyzed ina variety of systems. The compounds can be assessed in definedwell-known model systems used to assess cellular permeability, toxicity,and pharmacodynamic effects. These assays include both cell-based andanimal based assays.

Cell-Based Assay.

Cells from a P388 cell line (CellGate, Inc., Sunnyvale, Calif.) or humanmalignant melanoma cell line SK-MEL-2 are grown in RPMI 1640 cell mediumcontaining fetal calf serum (10%), L-glutamine, penicillin, streptomycinand are split twice weekly. All compounds are first diluted with DMSO.Later serial dilutions are done with a phosphate-buffered salinesolution. All dilutions are done in glass vials and the final DMSOconcentration is generally below 0.5% by volume. Final two-folddilutions are done in a 96-well plate using cell media so that each wellcontains 50 μL. All compounds are assayed over multiple concentrations.Cell concentration is measured using a hemacytometer and the final cellconcentration is adjusted to about 1×10⁴ cells/mL with cell medium. Theresulting solution of cells (50 μL) is then added to each well and theplates are incubated for 5 days in a 37° C., 5% CO₂, humidifiedincubator. MTT solution(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 10 μL) isthen added to each well and the plates are re-incubated under identicalconditions for 2 hours. To each well is then added acidified isopropanol(150 μL of i-PrOH solution containing 0.05 N HCl) and mixed thoroughly.The plates are then scanned at 595 nm and the absorbances are read(Wallac Victor 1420 Multilabel Counter). The resulting data is thenanalyzed to determine an ED₅₀ value. Compounds that kill cancer cells,but fail to kill normal cells, find application in the prevention ortreatment of cancer.

Mouse Ovarian Carcinoma Zenograft Model.

Compounds of the invention are evaluated in an ovarian carcinomaxenograft model of cancer, based on that described by Davis, et al.((1993) Cancer Research 53:2087-2091). This model, in brief, involvesinoculating female nu/nu mice with 1×10⁹ OVCAR3-icr cells into theperitoneal cavity. One or more test compounds are administered, e.g.,prior to or after tumor cell injection, by the oral route as asuspension in 1% methyl cellulose or intraperitoneally as a suspensionin phosphate-buffered saline in 0.01% TWEEN-20. At the conclusion of theexperiment (4-5 weeks) the number of peritoneal cells are counted andany solid tumor deposits weighed. In some experiments tumor developmentis monitored by measurement of tumor specific antigens.

Rat Mammary Carcinoma Model.

Compounds of the invention are evaluated in a HOSP.1 rat mammarycarcinoma model of cancer (Eccles, et al. (1995) Cancer Res.56:2815-2822). This model involves the intravenous inoculation of 2×10⁴tumor cells into the jugular vein of female CBH/cbi rats. One or moretest compounds are administered, e.g., prior to or after tumor cellinjection, by the oral route as a suspension in 1% methyl cellulose orintraperitoneally as a suspension in phosphate-buffered saline and 0.01%TWEEN-20. At the conclusion of the experiment (4-5 weeks) the animalsare killed, the lungs are removed and individual tumors counted after 20hours fixation in Methacarn.

Mouse B16 Melanoma Model.

The anti-metastatic potential of compounds of the invention is evaluatedin a B16 melanoma model in C57BL/6. Mice are injected intravenously with2×10⁵ B16/F10 murine tumor cells harvested from in vitro cultures.Inhibitors are administered by the oral route as a suspension in 1%methyl cellulose or intraperitoneally as a suspension inphosphate-buffered saline pH 7.2 and 0.01% TWEEN-20. Mice are killed 14days after cell inoculation and the lungs removed and weighed prior tofixing in Bouin's solution. The number of colonies present on thesurface of each set of lungs is then counted.

What is claimed is:
 1. A pharmaceutical composition comprising acompound having a structure of Formula IV in admixture with apharmaceutically acceptable carrier,

wherein R₉ and the dashed bond are either present or absent, whereinwhen R₉ is present R₉ is oxygen or carbon; R₁₀ and R₁₁ are independentlyhydrogen or a carboxyl group; R₁₂ and R₁₃ are independently —C(O)NH— or—NHC(O)—; and R₁₄ and R₁₅ are independently an unsubstitutedheterocyclic group or a benzene or heterocyclic group substituted withat least one substituent comprising a lower alkyl, amine, oxo, or—OC(O)CH₃.
 2. The pharmaceutical composition of claim 1, furthercomprising a cancer therapeutic agent.
 3. The pharmaceutical compositionof claim 2, wherein the cancer therapeutic agent comprises achemotherapeutic agent or radiotherapeutic agent.